ST2L antibody antagonists

ABSTRACT

The present invention relates to ST2L antagonists, polynucleotides encoding the antagonists or fragments thereof, and methods of making and using the foregoing.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/640,407, filed 30 Apr. 2012 and U.S. Provisional Application No.61/640,238, filed 30 Apr. 2012, the entire contents of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to ST2L antagonists, polynucleotidesencoding the antagonists or fragments thereof, and methods of making andusing the foregoing.

2. Description of Related Art

ST2L (IL-1RL1 or IL-33Rα) is a Toll/IL-1 receptor family memberexpressed on the cell surface of a wide variety of immune cellsincluding T cell, NK/NKT cells, basophils, eosinophils, mast cells andthe newly-described non-B/non-T innate lymphoid type 2 cells, nuocytes,and natural helper cells. ST2L expression is also inducible on dendriticcells (DCs), macrophages, and neutrophils. ST2L is able to downregulatethe responsiveness of Toll-like Receptors TLR2, TLR4, and TLR9, but alsoinduce type 2 cytokine release via activation by its ligand IL-33 andassociation with accessory protein IL-1RAcP. IL-33 has been described asan ‘alarmin’, as its full-length form resides in the nuclei ofepithelial and endothelial cells during homeostasis, but can be cleavedand released during necrosis.

ST2L signaling requires association of the accessory protein IL-1RAcP topreformed ST2L/IL-33 complex. The accessory protein IL-1RAcP is sharedwith the IL-1α/β signaling complex. Models of ST2L, IL-33, and IL-1RAcPinteractions as well as interactions between IL-1R1 and IL-1RAcP havebeen proposed (Lingel et al., Cell 17:1398-1410, 2009; Wang et al., NatImmunol 11:905-11, 2010). Recently, ST2L/IL-33/IL-1RAcP has been shownto form a signaling complex with c-Kit on mast cells, the receptor forstem cell factor (SCF). IL-33 induced cytokine production in primarymast cells in an SCF-dependent manner (Drube et al., Blood 115:3899-906,2010).

Activation of ST2L leads to excessive type 2 cytokine responses(especially IL-5 and IL-13), mast cell and eosinophil activation, andairway hyper-reactivity, and has also been reported to amplify Th1 andTh17 responses through induction of IFNγ from NKT cells and IL-1β andIL-6 from mast cells. Dysregulation of the ST2L/IL-33 pathway has beenimplicated in a variety of immune-mediated diseases, including asthma,rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis,allergic rhinitis, nasal polyposis, and systemic sclerosis (reviewed byPalmer and Gabay, Nat Rev Rheumatol 7:321-9, 2011 and Lloyd, Curr OpinImmunol 22:800-6, 2010; Shimizu et al., Hum Molec Gen 14:2919-27, 2005,Kamekura et al., Clin Exp Allergy 42:218-28, 2012; Manetti et al., AnnRheum Dis 69:598-605, 2010).

Thus, there is a need for ST2L antagonists that are suitable for use inthe treatment of ST2L mediated diseases and disorders.

BRIEF SUMMARY OF THE INVENTION

One aspect of the invention is an isolated human or human-adaptedantibody antagonist or fragment thereof that specifically binds Domain I(SEQ ID NO: 9) of human ST2L.

In another aspect, the invention provides for human or human-adaptedantibody antagonists specifically binding human ST2L having certainlight chain and heavy chain variable region sequences, or certain heavychain and light chain complementarity determining sequences.

In another aspect, the invention provides the invention provides forhuman or human-adapted antibody antagonists specifically biding humanST2L at defined epitope regions and/or having certain characteristics asdescribed herein.

Another aspect of the invention is an isolated polynucleotide encodingthe heavy chain variable regions (VH) or the light chain variableregions (VL) of the invention.

Another aspect of the invention is a vector comprising an isolatedpolynucleotide of the invention.

Another aspect of the invention is a host cell comprising a vector ofthe invention.

Another aspect of the invention is a method of producing an antibody ofthe invention comprising culturing a host cell of the invention andrecovering the antibody from the cell.

Another aspect of the invention is a pharmaceutical compositioncomprising an isolated antibody of the invention and a pharmaceuticallyaccepted carrier.

Another aspect of the invention is an method of treating or preventing aST2L-mediated condition comprising administering a therapeuticallyeffective amount of an isolated antibody of the invention to a patientin need thereof for a time sufficient to treat or prevent theST2L-mediated condition.

Another aspect of the invention is an method of inhibiting mast cellresponse in a patient comprising administering a therapeuticallyeffective amount of an isolated antibody of the invention to a patientin need thereof for a time sufficient to inhibit the mast cell response.

Another aspect of the invention is an method of inhibiting interactionof IL-33 and ST2L in a subject, comprising administering to the subjectan antibody that specifically binds domain I of ST2L in an amountsufficient to inhibit the interaction of IL-33 and ST2L.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows inhibition of airway hyper-responsiveness by ST2L Domain Ibinding mAb CNTO3914 in a model of lung inflammation induced byintranasally administered IL-33 when compared to the isotype controlCNTO5516. Peak airway resistance was measured upon methacholine (MCH)administration at increased doses (mg/ml). **p<0.05 for CNTO3914/IL-33vs. CNTO5516/IL-33; and ***p<0.001 for CNTO3914/IL-33 vs. PBS with IL-33treatment group.

FIG. 2 shows inhibition of Bronchoalveolar Lavage (BAL) cell recruitmentby ST2L Domain I binding mAb CNTO3914 in a model of lung inflammationinduced by intranasally administered IL-33 when compared to the isotypecontrol CNTO5516. ***p<0.001.

FIG. 3 shows dose-dependent inhibition of release of mouse Mast CellProtease 1 (MMCP-1) by ST2L Domain I binding mAb CNTO3914 in cell freeBAL fluid in a model of lung inflammation induced by intranasallyadministered IL-33. **p<0.01, ***p<0.001, vs. CNTO5516 (isotype control)with IL-33 treatment.

FIG. 4 shows inhibition of IL-33-induced GM-CSF (FIG. 4A), IL-5 (FIG.4B), and TNFα (FIG. 4C) release by ST2L Domain I binding mAb CNTO3914 bymouse bone marrow-derived mast cells in vitro. The CNTO3914concentrations used are shown as μg/ml and IL-33 concentrations as ng/mlin parenthesis.

FIG. 5 shows inhibition of IL-33-induced prostaglandin D2 (PGD₂) releaseby human cord blood-derived mast cells by ST2L Domain I binding mAbC2494 (STLM62) at indicated IL-33 and C2494 concentrations. MOX-PDG₂:methoxylsamine-PGD₂.

FIG. 6 shows inhibition of A) GM-CSF, B) IL-8, C) IL-5, D) IL-13 and E)IL-10 release by indicated concentrations (μg/ml) of ST2L Domain Ibinding mAbs C2244 and C2494 in human cord blood derived mast cells(hCBMCs) in the presence of 1 ng/ml IL-33 in StemPro-34 medium+100 ng/mlSCF (stem cell factor).

FIG. 7 shows effect on A) GM-CSF, B) IL-8, C) IL-5, D) IL-13 and E)IL-10 release by indicated concentrations (μg/ml) of ST2L Domain IIIbinding mAbs C2519 or C2521 in human cord blood-derived mast cell in thepresence of 1 ng/ml IL-33 in StemPro-34 medium+100 ng/ml SCF.

FIG. 8 shows effect on A) GM-CSF; B) IL-8; C) IL-5; D) IL-13 and E)IL-10 release by ST2L Domain I binding mAb C2494 and ST2L Domain IIIbinding mAbs ST2M48 (M48), ST2M49 (M49), ST2M50 (M50), and ST2M51 (M51)in human cord blood-derived mast cells (hCBMCs) in the presence of 3ng/ml IL-33 in RPMI/10% FCS medium+100 ng/ml SCF.

FIG. 9 shows average percent (%) inhibition of anti-ST2L antibodiesbinding Domain I (D1) or Domain III (D3) of ST2L on GM-CSF, IL-5, IL-8,IL-10 and IL-13 release by human cord blood-derived mast cells uponIL-33 and SCF induction as indicated using either 50 μg/ml or 2 μg/ml ofeach antibody tested. Negative values indicate % activation.

FIG. 10 shows heavy chain variable regions (VH) and heavy chain CDRsequences of anti-ST2L antibodies derived from phage display librariesand after subsequent affinity-maturation campaigns.

FIG. 11 shows light chain variable regions (VL) and light chain CDRsequences of anti-ST2L antibodies derived from phage display librariesand after subsequent affinity-maturation campaigns.

FIG. 12 shows VH and VL regions and sequences of heavy chain CDRs ofanti-ST2L antibody STLM208 VH ST2H257 HCDR3 variants.

FIG. 13 shows A) VH and B) VL sequences of anti-ST2L antibodies derivedfrom phage display libraries and after subsequent affinity-maturationcampaigns.

FIG. 14 shows delineation of C2494 VH and VL antigen binding sitestransferred to human frameworks (transferred marked as HFA, “humanframework adaptation”). Kabat CDRs are underlined and Chothia HVsindicated in dashed lines above the indicated transferred HFA regions.Numbering of VH and VL residues is according to Chothia. Residueshighlighted in grey in VH were not transferred in some HFA variants.C2494 VH: SEQ ID NO: 48; C2494 VH: SEQ ID NO: 52.

FIG. 15 shows CDR sequences of human framework adapted (HFA) antibodiesderived from C2494.

FIG. 16 A) Serum levels of anti-ST2L antibody CNTO3914 B) inhibition ofbronchoalveolar Lavage (BAL) cell recruitment C) inhibition of IL-6secretion by whole blood cells stimulated with IL-33; D) inhibition ofMCP1 secretion by whole blood cells stimulated with IL-33 by CNTO3914 24hours post-dosing in a 6 hour model of lung inflammation induced byintranasally administered IL-33. *p<0.05, **p<0.01, ***p<0.001; NQ=belowthe limit of detection; @=one data point is below the limit ofdetection.

FIG. 17. Competition between various anti-ST2L antibodies. A) 30 nMlabeled C2244 Fab was competed with indicated antibodies for binding toST2L-ECD coated on microwells. C2244 competed with C2494 but not withC2539. B) 10 nM labeled C2494 was competed with indicated antibodies forbinding to ST2L-ECD coated on microwells. C2494 competed with STLM208and STLM213 but not with C2539.

FIG. 18 shows a simplified H/D exchange map of the human ST2-ECD (SEQ IDNO: 119) complexed with C2244 Fab. The regions protected by the antibodywere displayed in different gray scale as indicated. Segmentsencompassing residues 18-31 (boxed in dashed line) (corresponding toresidues 35-48 of full length ST2L of SEQ ID NO: 1) were protected bythe Fab. Region encompassing residues 71-100 (boxed in solid line)(corresponding to residues 88-117 of SEQ ID NO: 1) were heavilyglycosylated and not covered by peptides.

FIG. 19 shows kinetic and affinity constants for ST2L Domain I bindingantibody for ST2L variants as indicated in the figure.

FIG. 20 shows inhibition of A) GM-CSF; B) IL-5; C) IL-8; D) IL-13secretion from primary human lung mast cells by an anti-ST2L antibodySTLM208.

DETAILED DESCRIPTION OF THE INVENTION

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as though fully set forth.

It is to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art to which the invention pertains.

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice for testing of the presentinvention, exemplary materials and methods are described herein. Indescribing and claiming the present invention, the following terminologywill be used.

The term “antagonist” as used herein means a molecule that partially orcompletely inhibits, by any mechanism, ST2L biological activity.Exemplary antagonists are antibodies, fusion proteins, peptides,peptidomimetics, nucleic acids, oligonucleotides and small molecules.Antagonists can be identified using assays for ST2L biological activitydescribed below. ST2L antagonists may inhibit measured ST2L biologicalactivity by 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or 100%.

The term “ST2L” or “huST2L” or “human ST2L” refers to a human ST2Lpolypeptide having an amino acid sequence shown in GenBank Acc. No.NP_(—)057316. SEQ ID NO: 1 shows the amino acid sequence of the fulllength human ST2L. “ST2L extracellular domain”, “ST2L-ECD” or“huST2L-ECD” as used herein means a polypeptide having amino acids19-328 of SEQ ID NO: 1. huST2L-ECD has three Ig-like C2-type domainsspanning residues 19-122 (Domain I, SEQ ID NO: 9), residues 123-202(Domain II, SEQ ID NO: 10), and residues 209-324 (Domain III, SEQ ID NO:11) of SEQ ID NO: 1. “Domain I” or “ST2L Domain I” or “huST2L Domain I”or “D1” refers to the first immunoglobulin-like domain on human ST2Lhaving the sequence shown in SEQ ID NO: 9. “Domain III” or “ST2L DomainIII” refers to the third immunoglobulin-like domain on human ST2L havingthe sequence shown in SEQ ID NO: 11.

The term “IL-33” as used herein includes full length IL-33 (GenBank Acc.No. NP_(—)254274 SEQ ID NO: 3), variants and active forms thereof. IL-33variants include proteins having amino acid sequences shown in GenBankAcc. No. NP_(—)001186569 and GenBank Acc. No. NP_(—)001186570). IL-33active forms include a “mature IL-33” having residues 112-270 of SEQ IDNO: 3. Additional active forms include IL-33 fragments having residues11-270, 115-270, 95-270, 99-270, or 109-270 of SEQ ID NO: 3 (LeFrancaiset al., Proc Natl Acad Sci (USA) 109:1673-8, 2012), or any form orcombination of forms isolated from cells endogenously expressing IL-33.“IL-33 active form” is a fragment or a variant of IL-33 of SEQ ID NO: 3that induces ST2L biological activity.

The term “antibodies” as used herein is meant in a broad sense andincludes immunoglobulin molecules including polyclonal antibodies,monoclonal antibodies including murine, human, human-adapted, humanizedand chimeric monoclonal antibodies, antibody fragments, bispecific ormultispecific antibodies formed from at least two intact antibodies orantibody fragments, dimeric, tetrameric or multimeric antibodies, andsingle chain antibodies.

Immunoglobulins can be assigned to five major classes, namely IgA, IgD,IgE, IgG and IgM, depending on the heavy chain constant domain aminoacid sequence. IgA and IgG are further sub-classified as the isotypesIgA₁, IgA₂, IgG₁, IgG₂, IgG₃ and IgG₄. Antibody light chains of anyvertebrate species can be assigned to one of two clearly distinct types,namely kappa (K) and lambda (A), based on the amino acid sequences oftheir constant domains.

The term “antibody fragments” refers to a portion of an immunoglobulinmolecule that retains the heavy chain and/or the light chain antigenbinding site, such as a heavy chain complementarity determining regions(HCDR) 1, 2 and 3, a light chain complementarity determining regions(LCDR) 1, 2 and 3, a heavy chain variable region (VH), or a light chainvariable region (VL). Antibody fragments include well known Fab,F(ab′)2, Fd and Fv fragments as well as a domain antibodies (dAb)consisting one VH domain. VH and VL domains can be linked together via asynthetic linker to form various types of single chain antibody designswhere the VH/VL domains pair intramolecularly, or intermolecularly inthose cases when the VH and VL domains are expressed by separate singlechain antibody constructs, to form a monovalent antigen binding site,such as single chain Fv (scFv) or diabody; described for example in Int.Pat. Publ. No. WO98/44001, Int. Pat. Publ. No. WO88/01649; Int. Pat.Publ. No. WO94/13804; Int. Pat. Publ. No. WO92/01047

An antibody variable region consists of a “framework” region interruptedby three “antigen binding sites”. The antigen binding sites are definedusing various terms: (i) Complementarity Determining Regions (CDRs),three in the VH (HCDR1, HCDR2, HCDR3), and three in the VL (LCDR1,LCDR2, LCDR3), are based on sequence variability (Wu and Kabat, J ExpMed 132:211-50, 1970; Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md., 1991). (ii) “Hypervariableregions”, “HVR”, or “HV”, three in the VH (H1, H2, H3) and three in theVL (L1, L2, L3), refer to the regions of an antibody variable domainswhich are hypervariable in structure as defined by Chothia and Lesk(Chothia and Lesk, Mol Biol 196:901-17, 1987). Other terms include“IMGT-CDRs” (Lefranc et al., Dev Comparat Immunol 27:55-77, 2003) and“Specificity Determining Residue Usage” (SDRU) (Almagro, Mol Recognit17:132-43, 2004). The International ImMunoGeneTics (IMGT) database(http://www_imgt_org) provides a standardized numbering and definitionof antigen-binding sites. The correspondence between CDRs, HVs and IMGTdelineations is described in Lefranc et al., Dev Comparat Immunol27:55-77, 2003.

“Chothia residues” as used herein are the antibody VL and VH residuesnumbered according to Al-Lazikani (Al-Lazikani et al., J Mol Biol273:927-48, 1997).

“Framework” or “framework sequences” are the remaining sequences of avariable region other than those defined to be antigen binding site.Because the antigen binding site can be defined by various terms asdescribed above, the exact amino acid sequence of a framework depends onhow the antigen-binding site was defined.

“Human antibody” or “fully human antibody” refers to antibodiescontaining variable region and constant region sequences derived fromhuman immunoglobulin sequences. Human antibodies of the invention mayinclude substitutions so that they may not be exact copies of expressedhuman immunoglobulin or germline gene sequences. However, antibodies inwhich antigen binding sites are derived from a non-human species are notincluded in the definition of “human antibody”.

“Human-adapted” antibodies or “human framework adapted (HFA)” antibodiesrefers to antibodies adapted according to methods described in U.S. Pat.Publ. No. US2009/0118127 and also refers to antibodies in whichantigen-binding site sequences derived from non-human species aregrafted onto human frameworks.

“Humanized antibodies” refers to antibodies wherein the antigen bindingsites are derived from non-human species and the variable regionframeworks are derived from human immunoglobulin sequences. Humanizedantibodies may include substitutions in the framework regions so thatthe framework may not be an exact copy of expressed human immunoglobulinor germline gene sequences.

The term “substantially identical” as used herein means that the twoantibody variable region amino acid sequences being compared areidentical or have “insubstantial differences”. Insubstantial differencesare substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids inan antibody or antibody variable region sequence that do not adverselyaffect antibody properties. Amino acid sequences substantially identicalto the variable region sequences disclosed herein are within the scopeof the application. In some embodiments, the sequence identity can beabout 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher.Percent identity can be determined for example by pairwise alignmentusing the default settings of the AlignX module of Vector NTI v.9.0.0(Invitrogen, Carslbad, Calif.). The protein sequences of the presentinvention can be used as a query sequence to perform a search againstpublic or patent databases to, for example, identify related sequences.Exemplary programs used to perform such searches are the XBLAST orBLASTP programs (http_//www_ncbi_nlm/nih_gov), or the GenomeQuest™(GenomeQuest, Westborough, Mass.) suite using the default settings.

The term “epitope” as used herein means a portion of an antigen to whichan antibody specifically binds. Epitopes usually consist of chemicallyactive (such as polar, non-polar or hydrophobic) surface groupings ofmoieties such as amino acids or polysaccharide side chains and can havespecific three-dimensional structural characteristics, as well asspecific charge characteristics. An epitope can be composed ofcontiguous and/or discontiguous amino acids that form a conformationalspatial unit. For a discontiguous epitope, amino acids from differingportions of the linear sequence of the antigen come in close proximityin 3-dimensional space through the folding of the protein molecule. Anexemplary epitope is Domain I of huST2L shown in SEQ ID NO: 9.

The term “paratope” as used herein means a portion of an antibody towhich an antigen specifically binds. A paratope can be linear in natureor can be discontinuous, formed by a spatial relationship betweennon-contiguous amino acids of an antibody rather than a linear series ofamino acids. A “light chain paratope” and a “heavy chain paratope” or“light chain paratope amino acid residues” and “heavy chain paratopeamino acid residues” refer to antibody light chain and heavy chainresidues in contact with an antigen, respectively.

The term “specific binding” or “specifically binds” as used hereinrefers to antibody binding to a predetermined antigen with greateraffinity than for other antigens or proteins. Typically, the antibodybinds to a predetermined antigen with a dissociation constant (K_(D)) of1×10⁻⁷ M or less, for example 1×10⁻⁸ M or less, 1×10⁻⁹ M or less,1×10⁻¹⁰ M or less, 1×10⁻¹¹ M or less, or 1×10⁻¹² M or less, typicallywith a K_(D) that is at least ten fold less than its K_(D) for bindingto a non-specific antigen (e.g., BSA, casein, or any other specifiedpolypeptide). The dissociation constant can be measured using standardprocedures. Antibodies that specifically bind to a predetermined antigenmay, however, have cross-reactivity to other related antigens, forexample to the same predetermined antigen from other species (homologs),such as human or monkey, for example Macaca fascicularis (cynomolgus).

“Bispecific” as used herein refers to an antibody that binds twodistinct antigens or two distinct epitopes within an antigen.

“Monospecific” as used herein refers to an antibody that binds oneantigen or one epitope.

The term “in combination with” as used herein means that the describedagents can be administered to an animal together in a mixture,concurrently as single agents or sequentially as single agents in anyorder.

“Inflammatory condition” as used herein refers to acute or chroniclocalized or systemic responses to harmful stimuli, such as pathogens,damaged cells, physical injury or irritants, that are mediated in partby the activity of cytokines, chemokines, or inflammatory cells (e.g.,neutrophils, monocytes, lymphocytes, macrophages) and is characterizedin most instances by pain, redness, swelling, and impairment of tissuefunction.

The term “ST2L-mediated inflammatory condition” as used herein refers toan inflammatory condition resulting at least partially frominappropriate activation of ST2L signaling pathway. ExemplaryST2L-mediated inflammatory conditions are asthma and allergies.

The term “ST2L-mediated condition” as used herein encompasses alldiseases and medical conditions in which ST2L plays a role, whetherdirectly or indirectly, in the disease or medical condition, includingthe causation, development, progress, persistence or pathology of thedisease or condition.

The term “ST2L biological activity” as used herein refers to anyactivity occurring as a result of ST2L ligand IL-33 binding to ST2L. Anexemplary ST2L biological activity results in activation of NF-κB inresponse to IL-33. NF-κB activation can be assayed using a reporter-geneassay upon induction of ST2L with IL-33 (Fursov et al., Hybridoma 30:153-62, 2011). Other exemplary ST2L biological activities result inproliferation of Th2 cells, or secretion of pro-inflammatory cytokinesand chemokines, for example IL-5, GM-CSF, IL-8, IL-10, or IL-13. Therelease of cytokines and chemokines from cells, tissues or incirculation can be measured using well-known immunoassays, such as anELISA immunoassay.

The term “vector” means a polynucleotide capable of being duplicatedwithin a biological system or that can be moved between such systems.Vector polynucleotides typically contain elements, such as origins ofreplication, polyadenylation signal or selection markers, that functionto facilitate the duplication or maintenance of these polynucleotides ina biological system. Examples of such biological systems may include acell, virus, animal, plant, and reconstituted biological systemsutilizing biological components capable of duplicating a vector. Thepolynucleotide comprising a vector may be DNA or RNA molecules or ahybrid of these.

The term “expression vector” means a vector that can be utilized in abiological system or in a reconstituted biological system to direct thetranslation of a polypeptide encoded by a polynucleotide sequencepresent in the expression vector.

The term “polynucleotide” means a molecule comprising a chain ofnucleotides covalently linked by a sugar-phosphate backbone or otherequivalent covalent chemistry. Double and single-stranded DNAs and RNAsare typical examples of polynucleotides.

The term “polypeptide” or “protein” means a molecule that comprises atleast two amino acid residues linked by a peptide bond to form apolypeptide. Small polypeptides of less than 50 amino acids may bereferred to as “peptides”.

Conventional one and three-letter amino acid codes are used herein asfollows:

Amino acid Three-letter code One-letter code Alanine ala A Arginine argR Asparagine asn N Aspartate asp D Cysteine cys C Glutamate glu EGlutamine gln Q Glycine gly G Histidine his H Isoleucine ile I Leucineleu L Lysine lys K Methionine met M Phenylalanine phe F Proline pro PSerine ser S Threonine thr T Tryptophan trp W Tyrosine tyr Y Valine valVCompositions of Matter

The present invention provides antibodies specifically binding ST2L andinhibiting ST2L biological activity, and uses of such antibodies. Theinventors have made a surprising finding that antibodies binding toDomain I of human ST2L (SEQ ID NO: 9) block IL-33/ST2L interaction andinhibit a spectrum of ST2L biological activities including IL-33-inducedmast cell responses, whereas antibodies binding Domain III of human ST2L(SEQ ID NO: 11) do not block IL-33/ST2L interaction although they areinhibitory in a spectrum of ST2L biological activities. Domain IIIbinding antibodies however have reduced or no inhibitory effect on, orin some cases stimulate IL-33-induced mast cell responses.

In some embodiments, the antibodies that block IL-33/ST2L interactionand inhibit a spectrum of ST2L biological activities includingIL-33-induced mast cell responses bind an epitope within human ST2LDomain I, (RCPRQGKPSYTVDW; SEQ ID NO: 210), and optionally ST2L aminoacid residues T93 and F94 (residue numbering according to SEQ ID NO: 1).

The term “mast cell response” or “mast cell activity” refers to theIL-33-induced release of cytokines such as GM-CSF, IL-8, IL-5, IL-13,and IL-10, and allergic mediators such as prostaglandin D₂ from mastcells.

The invention provides novel antigen-binding sites binding Domain I ofhuman ST2L. The structure for carrying an antigen-binding site istypically an antibody VH or VL.

One embodiment of the invention is an isolated human or human-adaptedantibody antagonist or fragment thereof that specifically binds Domain I(SEQ ID NO: 9) of human ST2L. An exemplary antibody binding Domain I ofhuman ST2L (SEQ ID NO: 9) is an antibody STLM15 (C2244) comprisingHCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 23, 27 and 31,respectively, and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 35, 39and 43, respectively, or an antibody C2494 (STLM62) comprising HCDR1,HCDR2 and HCDR3 sequences of SEQ ID NOs: 24, 28 and 32, respectively,and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 36, 40 and 44,respectively (Table 3). Additional exemplary antibodies binding Domain Iof human ST2L are antibodies shown in Table 16 and FIG. 13, for exampleantibodies STLM103, STLM107, STLM108, STLM123, STLM124, STLM208,STLM209, STLM210, STLM211, STLM212, and STLM213. Exemplary humanantibody antagonists are shown in FIG. 12 and FIG. 13. Exemplaryhuman-adapted antagonists are shown in Table 14.

In another embodiment, the isolated human or human-adapted antibodyantagonist or fragment thereof that specifically binds Domain I (SEQ IDNO: 9) of human ST2L blocks IL-33/ST2L interaction.

Antibodies can be tested for their ability to block IL-33/ST2Linteraction by standard ELISA. For example, plates are coated withextracellular domain of human ST2L (huST2L-ECD) and incubated with anantibody, after which binding of biotinylated IL-33 onto the plates ismeasured. Antibodies that “block IL-33/ST2L interaction” or “inhibitIL-33/ST2L interaction” are antibodies that in an ELISA assay usinghuST2L-ECD coated plates, reduce the signal derived from biotinylatedIL-33 bound to the plate by at least 30%, 40%, 50%, 60%, 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% at 50 μg/ml antibodyconcentration when compared to binding of IL-33 in the absence of theantibody.

Antibodies can be tested for their inhibition of mast cell responses byassessing their inhibitory activity on for example GM-CSF, IL-5, IL-10or IL-13 release by human cord blood-derived mast cells or primary humanlung mast cells using standard methods and methods exemplified infra.Antibodies that “inhibit mast cell response” or “inhibit mast cellactivity” are antibodies that reduce 1-3 ng/ml IL-33-induced GM-CSF,IL-5, IL-13 or IL-10 secretion by at least 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 960, 970, 980, 990 or 100% at aconcentration of 10 μg/ml when compared to mast cells not treated by theantibody. Typically mast cells may be derived from human cord blood orlung parenchyma and small airways CD34⁺ progenitors by well knownmethods and as exemplified infra. Mast cell culture conditions mayaffect the measure of % inhibition for an antibody and therefore cultureand test conditions may be kept standard using for example StemPro-34media throughout the 6-10 week long differentiation procedure. At 4 daysprior to the cytokine release assay mast cells are stimulated daily with10 ng/ml IL-4, 10 ng/ml IL-6 and 100 ng/ml SCF. For the cytokine releaseassay, mast cells can be resuspended in fresh StemPro-34 media or RPMIcontaining 10% FCS without antibiotics, with 100 ng/ml SCF. Suitableplating densities for assays are 65,000 to 75,000 cells/0.16 mls/well.Exemplary antibodies of the invention inhibiting mast cell responses areantibodies STLM15, STLM62 and STLM208. Antibody CNTO3914 binds mouseST2L Domain I without cross-reactivity to human ST2L and inhibits mastcell responses in mouse cells.

Those skilled in the art will appreciate that mast cell responses alsoinclude release of IL-1 and IL-32, and chemokines such as CCL1, CCL4,CCL5, CCL18 and CCL23 as well as allergic mediators such as cysteinylleukotrienes, histamine, as well as a variety of mast cell proteasesincluding tryptase, chymase, carboxypeptidase, and cathepsin G.Antibodies of the invention can be tested for their ability to inhibitthese additional mast cell responses using standard methods. Antibodiesof the invention binding Domain I of ST2L and blocking IL-33/ST2Linteraction can be expected to inhibit these additional mast cellresponses at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or more when tested at a minimum concentration of 10 μg/mlunder these conditions.

The antibodies of the invention bind human ST2L with a dissociationconstant (K_(D)) between about 5×10⁻¹² M to about 7×10⁻¹⁰ M, an on rateconstant (K_(on)) to human ST2L between about 2×10⁶ M⁻¹s⁻¹ to about1×10⁸ M⁻¹s⁻¹, or an off rate constant (K_(off)) to human ST2L betweenabout 1×10⁻⁶ s⁻¹ to about 1×10⁻² s⁻¹. For example, the antibodies of theinvention bind human ST2L with a K_(D) of less than about 7×10⁻¹⁰ M,less than about 1×10⁻¹⁰ M, less than about 5×10⁻¹¹ M, less than about1×10⁻¹¹ M or less than about 5×10⁻¹² M.

The antibodies of the invention may cross-react with Macaca fascicularis(cyno) ST2L (SEQ ID NO: 2) and bind to cyno ST2L with a dissociationconstant (K_(D)) between about 3×10⁻¹² M to about 2×10⁻⁹ M, an on rateconstant (K_(on)) to cyno ST2L between about 4×10⁶ M⁻¹s⁻¹ to about 1×10⁸M⁻¹s⁻¹, or an off rate constant (K_(off)) to cyno ST2L between about7×10⁻⁵ s⁻¹ to about 1×10⁻¹ s⁻¹. For example, the antibodies of theinvention bind cyno ST2L with a K_(D) of less than about 2×10⁻⁹ M, lessthan about 1×10⁻⁹ M, less than about 1×10⁻¹⁰ M, less than about 1×10⁻¹¹Mor less than about 3×10⁻¹² M.

The affinity of an antibody to ST2L can be determined experimentallyusing any suitable method. Such methods may utilize ProteOn XPR36,Biacore 3000 or KinExA instrumentation, ELISA or competitive bindingassays known to those skilled in the art. The measured affinity of aparticular antibody/ST2L interaction can vary if measured underdifferent conditions (e.g., osmolarity, pH). Thus, measurements ofaffinity and other binding parameters (e.g., K_(D), K_(on), K_(off)) arepreferably made with standardized conditions and a standardized buffer,such as the buffer described herein. Skilled in the art will appreciatethat the internal error for affinity measurements for example usingBiacore 3000 or ProteOn (measured as standard deviation, SD) cantypically be within 5-33% for measurements within the typical limits ofdetection. Therefore the term “about” reflects the typical standarddeviation in the assay. For example, the typical SD for a K_(D) of1×10⁻⁹ M is up to ±0.33×10⁻⁹ M.

The antibodies binding human ST2L with a desired affinity and optionallycross-reacting with cyno ST2L can be selected from libraries of variantsor fragments by panning with human and/or cyno ST2L and optionally byfurther antibody affinity maturation. Antibodies can be identified basedon their inhibition of ST2L biological activity using any suitablemethod. Such methods may utilize reporter-gene assays or assaysmeasuring cytokine production using well known methods and as describedin the application.

One embodiment of the invention is an isolated antibody antagonistspecifically binding human ST2L comprising:

-   -   a heavy chain complementarity determining region (HCDR) 1        (HCDR1) of SEQ ID NO: 160 (X₁X₂X₃MX₄); wherein        -   X₁ is S, F, D, I, G or V;        -   X₂ is Y or D;        -   X₃ is A, D or S; and        -   X₄ is S, F or I;    -   a HCDR 2 (HCDR2) of SEQ ID NO: 161 (X₅IX₆GX₇GGX₈TX₉YADSVKG);        wherein        -   X₅ is A, S, T, Y or D;        -   X₆ is S, R, E, K, G or A;        -   X₇ is S, E or N;        -   X₈ is S, R, E, G, T, D or A; and        -   X₉ is Y, D, N, A or S; and    -   a HCDR 3 (HCDR3) of SEQ ID NO: 162 (X₁₀X₁₁WSTEGSFFVLDY); wherein        -   X₁₀ is D, A, R, N, Q, P, E, I, H, S, T or Y; and        -   X₁₁ is P, A, H, Y, E, Q, L, S, N, T, V, or I.

Another embodiment of the invention is an isolated antibody antagonistspecifically binding human ST2L comprising:

-   -   a light chain complementarity determining region (LCDR) 1        (LCDR1) of SEQ ID NO: 163 (RASQSVDDX₁₂LA); wherein        -   X₁₂ is A or D;    -   a LCDR 2 (LCDR2) of SEQ ID NO: 90 (DASNRAT); and    -   a LCDR 3 (LCDR3) of SEQ ID NO: 164 (QQX₁₃X₁₄X₁₅X₁₆X₁₇X₁₈T);        wherein        -   X₁₃ is F or Y;        -   X₁₄ is Y, I or N;        -   X₁₅ is N, G, D or T;        -   X₁₆ is W or A;        -   X₁₇ is P or deleted; and        -   X₁₈ is L or I.

The antibodies of the invention comprising the HCDR1, HCDR2, HCDR3,LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 160, 161, 162, 163, 90and 164, respectively, can be made by well known mutagenesis methodsusing for example HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequencesof SEQ ID NOs: 78, 81, 84, 87, 90 and 92, respectively as a template.The heavy chain CDRs and the light chain CDRs of SEQ ID NOs: 160, 161,162, 163, 90 and 164, respectively, can be grafted to human frameworks,such as frameworks described infra. The antibodies can be assayed forbinding to ST2L and for their ability to block IL-33/ST2L interactionand for other characteristics such as affinity to human ST2L and/or cynoST2L, and inhibition of mast cell responses using methods describedherein.

In one embodiment, an isolated antibody antagonist specifically bindinghuman ST2L comprises:

the HCDR1 of SEQ ID NOs: 78 or 95-108;

the HCDR2 of SEQ ID NOs: 81, 109-118 or 120-129;

the HCDR3 of SEQ ID NOs: 84 or 165-185;

the LCDR1 of SEQ ID NOs: 87 or 130;

the LCDR2 or SEQ ID NO: 90; and

the LCDR3 of SEQ ID NOs: 92 or 131-134.

In another embodiment, an isolated antibody antagonist specificallybinding human ST2L comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3sequences as shown in FIG. 10, FIG. 11, and FIG. 12.

In another embodiment, an isolated antibody antagonist specificallybinding human ST2L comprises:

the HCDR1 of SEQ ID NOs: 23 or 24;

the HCDR2 of SEQ ID NOs: 27 or 28;

the HCDR3 of SEQ ID NOs: 31 or 32;

the LCDR1 of SEQ ID NOs: 35 or 36;

the LCDR2 or SEQ ID NOs: 39 or 40; and

the LCDR3 of SEQ ID NOs: 43 or 44.

In another embodiment, an isolated antibody antagonist specificallybinding human ST2L comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3sequences:

-   -   SEQ ID NOs: 23, 27, 31, 35, 39 and 43, respectively;    -   SEQ ID NOs: 24, 28, 32, 36, 40 and 44, respectively; (HFA CDRs);    -   SEQ ID NOs: 24, 28, 146, 36, 40 and 147, respectively; or    -   SEQ ID NOs: 24, 28, 146, 36, 40 and 44, respectively.

Another embodiment of the invention is an isolated human orhuman-adapted antibody antagonist or fragment thereof specificallybinding human ST2L (SEQ ID NO: 1) comprising a heavy chain variableregion (VH) comprising a VH framework derived from human IGHV3-23 (SEQID NO: 158), IGHV1-24*01 (SEQ ID NO: 148) or IGHV1-f*01 (SEQ ID NO: 149)framework sequences, and a light chain variable region (VL) comprising aVL framework derived from a human IGKV3-11 (L6) (SEQ ID NO: 159),IGKV3-15*01 (L2) (SEQ ID NO: 150), IGKV1-9*01 (L8) (SEQ ID NO: 151),IGKV1-5*01 (L12) (SEQ ID NO: 152), IGKV1-12*01 (L5) (SEQ ID NO: 153),IGKV1-39*01 (012) (SEQ ID NO: 154), IGKV1-27*01 (A20) (SEQ ID NO: 155)or IGKV1-33*01 (018) (SEQ ID NO: 156) framework sequences.

In another embodiment, the isolated antibody specifically binding humanDomain I of human ST2L comprises a VH comprising a VH framework derivedfrom human VH 3-23 framework sequences (SEQ ID NO: 158); and a lightchain variable region (VL) comprising a VL framework derived from ahuman Vκ L6 framework sequences (SEQ ID NO: 159). Human frameworksequences are well known, and typically include human immunoglobulingermline variable region sequences joined to the joining (J) sequences.The human VH 3-23 framework amino acid sequence shown in SEQ ID NO: 158includes human germline VH 3-23 sequence joined to IGHJ4 and the humanVk L6 framework amino acid sequence shown in SEQ ID NO: 159 includeshuman Vκ L6 germline sequence joined to IGKJ1 as described in Shi etal., J Mol Biol 397:385-96, 2010; Int. Pat. Publ. No. WO2009/085462; andU.S. Pat. Publ. No. US2010/0021477. Exemplary antibodies having a VHsequence derived from human VH 3-23 and a VL sequence derived from humanVκ L6 are those shown in FIG. 12 and FIG. 13.

Human or human-adapted antibodies comprising heavy or light chainvariable regions “derived from” a particular framework or germlinesequence refer to antibodies obtained from a system that uses humangermline immunoglobulin genes, such as from transgenic mice or fromphage display libraries as discussed infra. An antibody that is “derivedfrom” a particular framework or germline sequence may contain amino aciddifferences as compared to the sequence it was derived from, due to, forexample, naturally-occurring somatic mutations or intentionalsubstitutions.

In another embodiment, the isolated human or human-adapted antibodyantagonist or fragment thereof that specifically binds Domain I (SEQ IDNO: 9) of human ST2L competes for binding to human ST2L (SEQ ID NO: 1)with an isolated antibody comprising a heavy chain variable region (VH)of SEQ ID NO: 47 and a light chain variable region (VL) of SEQ ID NO: 51(antibody C2244).

In another embodiment, the isolated antibody of the invention bindshuman ST2L at amino acid residues 35-48 of SEQ ID NO: 1 (RCPRQGKPSYTVDW;SEQ ID NO: 210). The antibody may further bind human ST2L at amino acidresidues T93 and F94 of SEQ ID NO: 1.

Competition between specific binding to human ST2L with antibodies ofthe invention comprising certain HCDR1, HCDR2 and HCDR3, and LCDR1,LCDR2 and LCDR3 amino acid sequences or comprising certain VH and VLsequences can be assayed in vitro using well known methods. For example,binding of MSD Sulfo-Tag™ NHS-ester-labeled antibody to human ST2L inthe presence of an unlabeled antibody can be assessed by ELISA, orBiacore analyses or flow cytometry may be used to demonstratecompetition with the antibodies of the current invention. The ability ofa test antibody to inhibit the binding of C2244 to human ST2Ldemonstrates that the test antibody can compete with these antibodiesfor binding to human ST2L. Such exemplary antibodies are C2494, STLM208and STLM213 shown in Table 3 and FIG. 13.

Antibodies competing with C2244 for binding to Domain I of ST2L blockIL-33/ST2L interaction and inhibit a spectrum of ST2L biologicalactivities, including IL-33-induced mast cell responses. Anon-neutralizing (i.e. non-inhibiting) epitope is also present on ST2LDomain I, as a second antibody competition group (represented byantibody C2240 which binds Domain I of ST2L, does not compete withC2244, and does not inhibit ST2L signaling).

Antibodies of the invention binding specific ST2L domains or epitopescan be made by immunizing mice expressing human immunoglobulin loci(Lonberg et al., Nature 368:856-9, 1994; Fishwild et al., NatureBiotechnology 14:845-51, 1996; Mendez et al., Nature Genetics 15:146-56,1997, U.S. Pat. Nos. 5,770,429, 7,041,870, and 5,939,598) or Balb/c micewith the peptides encoding the epitopes, for example a peptide having anamino acid sequence of Domain I of human ST2L:KFSKQSWGLENEALIVRCPRQGKPSYTVDWYYSQTNKSIPTQERNRVFASGQLLKFLPAAVADSGIYTCIVRSPTFNRTGYANVTIYKKQSDCNVPDYLMYSTV (SEQ ID NO: 9), or a peptidehaving an amino acid sequence of RCPRQGKPSYTVDW (SEQ ID NO: 210), andusing the hybridoma method of Kohler et al., Nature 256:495-97, 1975.The resulting antibodies are tested for their binding to the epitopeusing standard methods. For example, when the structures of bothindividual components are known, in silico protein-protein docking canbe carried out to identify compatible sites of interaction.Hydrogen-deuterium (H/D) exchange can be carried out with the antigenand antibody complex to map regions on the antigen that may be bound bythe antibody. Segment and point mutagenesis of the antigen can be usedto locate amino acids important for antibody binding. The identifiedmAbs can further be modified by incorporating altered framework supportresidues to preserve binding affinity by techniques such as thosedisclosed in Queen et al., Proc Natl Acad Sci (USA) 86:10029-32, 1989and Hodgson et al., Bio/Technology 9:421, 1991.

The antibodies of the invention may be human or human-adapted. Theantibodies of the invention may be of IgA, IgD, IgE, IgG or IgM type.

Antibodies whose antigen-binding site amino acid sequences aresubstantially identical to those shown in FIG. 10, FIG. 11, FIG. 12,FIG. 13, FIG. 15, Table 3, Table 9 and Table 12 are encompassed withinthe scope of the invention. Typically, this involves one or more aminoacid substitutions with an amino acid having similar charge orhydrophobic or stereochemical characteristics, and are made to improveantibody properties, for example stability or affinity. For example, aconservative substitution may involve a substitution of a native aminoacid residue with a normative residue such that there is little or noeffect on the polarity or charge of the amino acid residue at thatposition. Furthermore, any native residue in the polypeptide may also besubstituted with alanine, as has been previously described for alaninescanning mutagenesis (MacLennan et al., Acta Physiol Scand Suppl643:55-67, 1998; Sasaki et al., Adv Biophys 35:1-24, 1998). Desiredamino acid substitutions (whether conservative or non-conservative) canbe determined by those skilled in the art at the time such substitutionsare desired. For example, amino acid substitutions can be used toidentify residues important for the function of the antibodies, such asresidues affecting affinity, or residues that impart undesirableproperties such as aggregation. Exemplary amino acid substitutions areshown in FIG. 12 and FIG. 13.

Substitutions in the framework regions, in contrast to antigen bindingsites may also be made as long as they do not adversely affect theproperties of the antibody. Framework substitutions can be made forexample at the Vernier Zone residues (U.S. Pat. No. 6,649,055) toimprove antibody affinity or stability. Substitutions can also be madeat those framework positions in the antibody that differ in sequencewhen compared to the homologous human germline gene sequences to reducepossible immunogenicity. These modifications can be done for example toantibodies derived from de novo antibody libraries, such as pIXlibraries.

Conservative amino acid substitutions also encompass non-naturallyoccurring amino acid residues which are typically incorporated bychemical peptide synthesis rather than by synthesis in biologicalsystems. Amino acid substitutions can be done for example by PCRmutagenesis (U.S. Pat. No. 4,683,195). Libraries of variants can begenerated using well known methods, for example using random (NNK) ornon-random codons, for example DVK codons, which encode 11 amino acids(ACDEGKNRSYW), and screening the libraries or variants with desiredproperties.

Although the embodiments illustrated in the Examples comprise pairs ofvariable regions, pairs of full length antibody chains, or pairs ofCDR1, CDR2 and CDR3 regions, one from a heavy chain and one from a lightchain, a skilled artisan will recognize that alternative embodiments maycomprise single heavy chain variable regions or single light chainvariable regions, single full length antibody chains, or CDR1, CDR2 andCDR3 regions from one antibody chain, either heavy or light. The singlevariable region, full length antibody chain or CDR1, CDR2 and CDR3region of one chain can be used to screen for corresponding domains inanother chain, the two chains capable of forming an antibody thatspecifically binds ST2L. The screening may be accomplished by phagedisplay screening methods using, e.g., a hierarchical dual combinatorialapproach disclosed in PCT Publ. No. WO1992/01047. In this approach, anindividual colony containing either a H or L chain clone is used toinfect a complete library of clones encoding the other chain (L or H),and the resulting two-chain specific antigen-binding domain is selectedin accordance with phage display techniques as described.

The invention provides for isolated VH and VL domains of the antibodiesof the invention and antibodies comprising certain VH and VL domains. VHand VL variable regions for certain antibodies of the invention areshown in FIG. 13 and Table 12.

One embodiment of the invention is an isolated human or human-adaptedantibody antagonist or fragment thereof that specifically binds Domain I(SEQ ID NO: 9) of human ST2L comprising the VH at least 90% identical tothe VH of SEQ ID NO: 191.

Another embodiment of the invention is an isolated human orhuman-adapted antibody antagonist or fragment thereof that specificallybinds Domain I (SEQ ID NO: 9) of human ST2L comprising the VL at least94% identical to the VL of SEQ ID NO: 209.

In one embodiment, the invention provides for an antibody comprising theVH of SEQ ID NOs: 143, 144, 145, 186, 187, 188, 189, 190, 191, 192, 193,194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204 or 205.

In another embodiment, the invention provides for an antibody comprisingthe VL of SEQ ID NOs: 135, 136, 137, 138, 139, 140, 141, 142, 206, 207,208 or 209.

In another embodiment, the invention provides for an antibody comprising

-   -   the VH of SEQ ID NOs: 186, 187, 197, 198, 199, 200, 201, 202,        203, 204 or 205 and the VL of SEQ ID NO: 206;    -   the VH of SEQ ID NOs: 195 or 196 and the VL of SEQ ID NO: 207;    -   the VH of SEQ ID NOs: 188, 189 or 190 and the VL of SEQ ID NO:        208; or    -   the VH of SEQ ID NOs: 187, 191, 192, 193 or 194 and the VL of        SEQ ID NO: 209.

Another embodiment of the invention an isolated human or human-adaptedantibody antagonist or fragment thereof that specifically binds Domain I(SEQ ID NO: 9) of human ST2L comprising:

the HCDR1 of SEQ ID NO: 97;

the HCDR2 of SEQ ID NO: 114;

the HCDR3 of SEQ ID NO: 84;

the LCDR1 of SEQ ID NO: 130;

the LCDR2 of SEQ ID NO: 90;

the LCDR3 of SEQ ID NO: 134; or

the VH of SEQ ID NO: 191 and the VL of SEQ ID NO: 209.

Human mAbs lacking any non-human sequences can be prepared and optimizedfrom phage display libraries by techniques referenced in, e.g., Knappiket al., J Mol Biol 296:57-86, 2000; and Krebs et al., J Immunol Meth254:67-84 2001. In an exemplary method, the antibodies of the inventionare isolated from libraries expressing antibody heavy and light chainvariable regions as fusion proteins with bacteriophage pIX coat protein.The antibody libraries are screened for binding to human ST2L-ECD andthe obtained positive clones are further characterized, the Fabsisolated from the clone lysates, and expressed as full length IgGs.Exemplary antibody libraries and screening methods are described in Shiet al., J Mol Biol 397:385-96, 2010; Int. Pat. Publ. No. WO2009/085462,and U.S. Ser. No. 12/546,850; U.S. Pat. Nos. 5,223,409, 5,969,108, and5,885,793).

The resulting mAbs can further be modified in their framework regions tochange certain framework residues to those present in a matching humangermline.

Immune effector properties of the antibodies of the invention may beenhanced or silenced through Fc modifications by techniques known tothose skilled in the art. For example, Fc effector functions such as Clqbinding, complement dependent cytotoxicity (CDC), antibody-dependentcell-mediated cytotoxicity (ADCC), phagocytosis, down regulation of cellsurface receptors (e.g., B cell receptor; BCR), etc. can be providedand/or controlled by modifying residues in the Fc responsible for theseactivities. Pharmacokinetic properties could also be enhanced bymutating residues in the Fc domain that extend antibody half-life(Strohl Curr Opin Biotechnol 20:685-91, 2009). Exemplary Fcmodifications are IgG4 S228P/L234A/L235A, IgG2 M252Y/S254T/T256E(Dall'Acqua et al., J Biol Chem 281:23514-24, 2006; or IgG2V234A/G237A/P238S, V234A/G237A/H268Q, H268A/V309L/A330S/P331 orV234A/G237A/P238S/H268A/V309L/A330S/P331S on IgG2 (Intl. Pat. Appl. No.WO2011/066501) (numbering according to the EU numbering).

Additionally, antibodies of the invention can be post-translationallymodified by processes such as glycosylation, isomerization,deglycosylation or non-naturally occurring covalent modification such asthe addition of polyethylene glycol moieties (pegylation) andlipidation. Such modifications may occur in vivo or in vitro. Forexample, the antibodies of the invention can be conjugated topolyethylene glycol (PEGylated) to improve their pharmacokineticprofiles. Conjugation can be carried out by techniques known to thoseskilled in the art. Conjugation of therapeutic antibodies with PEG hasbeen shown to enhance pharmacodynamics while not interfering withfunction (Knigh et al., Platelets 15:409-18, 2004; Leong et al.,Cytokine 16:106-19, 2001; Yang et al., Protein Eng 16:761-70, 2003).

Antibodies or fragments thereof of the invention modified to improvestability, selectivity, cross-reactivity, affinity, immunogenicity orother desirable biological or biophysical property are within the scopeof the invention. Stability of an antibody is influenced by a number offactors, including (1) core packing of individual domains that affectstheir intrinsic stability, (2) protein/protein interface interactionsthat have impact upon the HC and LC pairing, (3) burial of polar andcharged residues, (4) H-bonding network for polar and charged residues;and (5) surface charge and polar residue distribution among other intra-and inter-molecular forces (Worn et al., J Mol Biol 305:989-1010, 2001).Potential structure destabilizing residues may be identified based uponthe crystal structure of the antibody or by molecular modeling incertain cases, and the effect of the residues on antibody stability canbe tested by generating and evaluating variants harboring mutations inthe identified residues. One of the ways to increase antibody stabilityis to raise the thermal transition midpoint (Tm) as measured bydifferential scanning calorimetry (DSC). In general, the protein Tm iscorrelated with its stability and inversely correlated with itssusceptibility to unfolding and denaturation in solution and thedegradation processes that depend on the tendency of the protein tounfold (Remmele et al., Biopharm 13:36-46, 2000). A number of studieshave found correlation between the ranking of the physical stability offormulations measured as thermal stability by DSC and physical stabilitymeasured by other methods (Gupta et al., AAPS PharmSci 5E8, 2003; Zhanget al., J Pharm Sci 93:3076-89, 2004; Maa et al., Int J Pharm140:155-68, 1996; Bedu-Addo et al., Pharm Res 21:1353-61, 2004; Remmeleet al., Pharm Res 15:200-8, 1997). Formulation studies suggest that aFab Tm has implication for long-term physical stability of acorresponding mAb. Differences in amino acids in either framework orwithin the CDRs could have significant effects on the thermal stabilityof the Fab domain (Yasui et al., FEBS Lett 353:143-6, 1994).

Antibodies of the invention specifically binding Domain I of human ST2Lcan be engineered into bispecific antibodies which are also encompassedwithin the scope of the invention. The VL and/or the VH regions of theantibodies of the invention can be engineered using published methodsinto single chain bispecific antibodies as structures such as Tand Ab®designs (Int. Pat. Publ. No. WO1999/57150; U.S. Pat. Publ. No.US2011/0206672) or into bispecific scFVs as structures such as thosedisclosed in U.S. Pat. No. 5,869,620; Int. Pat. Publ. No. WO1995/15388A,int. Pat. Publ. No. WO1997/14719 or Int. Pat. Publ. No WO2011/036460.

The VL and/or the VH regions of the antibodies of the invention can beengineered into bispecific full length antibodies, where each antibodyarm binds a distinct antigen or epitope. Such bispecific antibodies aretypically made by modulating the CH3 interactions between the twoantibody heavy chains to form bispecific antibodies using technologiessuch as those described in U.S. Pat. No. 7,695,936; Int. Pat. Publ. No.WO04/111233; U.S. Pat. Publ. No. US2010/0015133; U.S. Pat. Publ. No.US2007/0287170; Int. Pat. Publ. No. WO2008/119353; U.S. Pat. Publ. No.US2009/0182127; U.S. Pat. Publ. No. US2010/0286374; U.S. Pat. Publ. No.US2011/0123532; Int. Pat. Publ. No. WO2011/131746; Int. Pat. Publ. No.WO2011/143545; or U.S. Pat. Publ. No. US2012/0149876. Additionalbispecific structures into which the VL and/or the VH regions of theantibodies of the invention can be incorporated are for example DualVariable Domain Immunoglobulins (Int. Pat. Publ. No. WO2009/134776), orstructures that include various dimerization domains to connect the twoantibody arms with different specificity, such as leucine zipper orcollagen dimerization domains (Int. Pat. Publ. No. WO2012/022811, U.S.Pat. No. 5,932,448; U.S. Pat. No. 6,833,441).

Another aspect of the invention is an isolated polynucleotide encodingany of the antibody heavy chain variable regions or the antibody lightchain variable regions or fragments thereof of the invention or theircomplement. Certain exemplary polynucleotides are disclosed herein,however, other polynucleotides which, given the degeneracy of thegenetic code or codon preferences in a given expression system, encodethe antibody antagonists of the invention are also within the scope ofthe invention. Exemplary polynucleotides of the invention are thoseshown in SEQ ID NOs: 211, 212, 213 and 214.

Another embodiment of the invention is a vector comprising thepolynucleotide of the invention. Such vectors may be plasmid vectors,viral vectors, vectors for baculovirus expression, transposon basedvectors or any other vector suitable for introduction of thepolynucleotides of the invention into a given organism or geneticbackground by any means.

Another embodiment of the invention is a host cell comprising thepolynucleotide of the invention. Such host cells may be eukaryoticcells, bacterial cells, plant cells or archeal cells. Exemplaryeukaryotic cells may be of mammalian, insect, avian or other animalorigins. Mammalian eukaryotic cells include immortalized cell lines suchas hybridomas or myeloma cell lines such as SP2/0 (American Type CultureCollection (ATCC), Manassas, Va., CRL-1581), NS0 (European Collection ofCell Cultures (ECACC), Salisbury, Wiltshire, UK, ECACC No. 85110503), FO(ATCC CRL-1646) and Ag653 (ATCC CRL-1580) murine cell lines. Anexemplary human myeloma cell line is U266 (ATTC CRL-TIB-196). Otheruseful cell lines include those derived from Chinese Hamster Ovary (CHO)cells such as CHO-K1SV (Lonza Biologics, Walkersville, Md.), CHO-K1(ATCC CRL-61) or DG44.

Another embodiment of the invention is a method of producing an antibodythat specifically binds Domain I of ST2L, comprising culturing a hostcell of the invention and recovering the antibody produced by the hostcell. Methods of making antibodies and purifying them are well known inthe art.

Another embodiment of the invention of a method of inhibitinginteraction of ST2L with IL-33 in a subject comprising administering thesubject an antibody specifically binding domain I of ST2L in an amountsufficient to inhibit interaction of ST2L and IL-33.

Methods of Treatment

ST2L antagonists of the invention, for example ST2L antibody antagonistsblocking IL-33/ST2L interaction and binding Domain I of ST2L, antibodiesthat compete for binding to human ST2L (SEQ ID NO: 1) with an isolatedantibody comprising a heavy chain variable region of SEQ ID NO: 47 and alight chain variable region of SEQ ID NO: 51, or antibodies bindinghuman ST2L at amino acid residues 35-48 of SEQ ID NO: 1 (RCPRQGKPSYTVDW;SEQ ID NO: 210) may be utilized to modulate the immune system.Antibodies of the invention may be more efficient in antagonizing ST2Lbiological activity when compared to antibodies binding other domainsand/or regions on ST2L as the antibodies of the invention are able tomore efficiently reduce IL-33-induced mast cell responses. Anyantibodies of the invention can be used in the methods of the invention.Exemplary antibodies that can be used in the methods of the inventionare antibodies STLM62, STLM15, STLM103, STLM107, STLM108, STLM123,STLM124, STLM206, STLM207, STLM208, STLM209, STLM210, STLM211, STLM212,STLM213. Without wishing to be bound by any theory, it is suggested thatantibody antagonists that bind Domain I and block IL-33/ST2L interactionmay inhibit formation of the IL-1RAcP/IL-33/ST2L/cKit complex ordownstream signaling on mast cells, whereas Domain III bindingantibodies, while being able to inhibit recruitment of IL-1RAcP to theST2L/IL-33 complex, may be unable to disrupt the formation of the largerIL-1RAcP/IL-33/ST2L/cKit complex specifically found on mast cells.Microarray analysis conducted supports the suggestion as it wasdemonstrated that anti-ST2L Domain I binding antibodies suppressed themajority of mast cell signaling pathways induced by IL-33, and thatanti-ST2L Domain III binding antibodies were only able to inhibit asmall subset of these signaling pathways. It is feasible that becauseIL-33 binds to ST2L prior to the association of the accessory proteinIL-1RAcP, blockade of IL-33 binding to ST2L by Domain I-bindingantibodies could prevent association of any other accessory protein,including cKit or as-yet unidentified co-receptors. Domain III-bindingantibodies, which do not interfere with IL-33 binding to ST2L, couldtheoretically block IL-1RAcP association but not the association ofother co-receptors, including as-yet unidentified co-receptors. Multiplemodels have been proposed for how IL-1RAcP interacts with the IL-1/IL-1Ror ST2L/IL-33 complexes (Lingel et al., Structure 17: 1398-1410, 2009;and reviewed by Thomas et al., Nat Struct & Molec Biol 19: 455-457,2012). These models indicate that IL-1RAcP could bind to one side of thecomplex, but which side has not been conclusively shown. Therefore it isfeasible that the ‘other side’ or ‘free side’ of the complex isavailable for association with an alternate co-receptor, that would notbe blocked by a Domain III antibody, and off-target effects such asincreased recruitment of another co-receptor, resulting in increasedsignaling, is possible.

In the methods of the invention, any antibody antagonist specificallybinding Domain I of human ST2L, antibody antagonist blocking IL-33/ST2Linteraction and binding Domain I of human ST2L, antibodies that competesfor binding to human ST2L (SEQ ID NO: 1) with an isolated antibodycomprising a heavy chain variable region of SEQ ID NO: 47 and a lightchain variable region of SEQ ID NO: 51, or antibodies binding human ST2Lat amino acid residues 35-48 of SEQ ID NO: 1 (RCPRQGKPSYTVDW; SEQ ID NO:210) may be used. Additional characteristics of such antibodies includeability of the antibody to block IL-33/ST2L interaction and to inhibithuman mast cell responses.

Therefore, antibodies of the invention are suitable for treating aspectrum of ST2L-mediated conditions, ST2L-mediated inflammatoryconditions and conditions where inhibition of mast cell responses isdesired.

The methods of the invention may be used to treat an animal patientbelonging to any classification. Examples of such animals includemammals such as humans, rodents, dogs, cats and farm animals. Forexample, the antibodies of the invention are useful in the prophylaxisand treatment of ST2L-mediated conditions, such as inflammatory diseasesincluding asthma, airway hyper-reactivity, sarcoidosis, chronicobstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis(IPF), cystic fibrosis, inflammatory bowel disease (IBD), rheumatoidarthritis, eosinophilic esophagitis, scleroderma, atopic dermatitis,allergic rhinitis, bullous pemphigoid, chronic urticaria, diabeticnephropathy, interstitial cystitis or Graft Versus Host Disease (GVHD).The antibodies of the invention are useful in the prophylaxis andtreatment of immune diseases mediated at least in part by mast cells,such as asthma, eczema, itch, allergic rhinitis, allergicconjunctivitis, as well as autoimmune diseases such as rheumatoidarthritis, bullous pemphigoid and multiple sclerosis.

The antibodies of the invention and are also useful in the preparationof a medicament for such treatment, wherein the medicament is preparedfor administration in dosages defined herein.

Mast cells play a central role in allergic inflammation and asthmathrough their release of a variety of mediators (reviewed by Amin,Respir Med 106:9-14, 2012). ST2L is highly expressed on mast cells andits activation leads to expression of many proinflammatory cytokines andother mediators. Inhibition of ST2L activity is proposed to interferewith mast cell mediated inflammatory cell recruitment and to modulatechronic inflammation.

Mast cells are rapid responders to stimulation, including allergen, coldair, pathogen; damage to the epithelium by these stimuli can result inrelease of IL-33 (reviewed by Zhao and Hu, Cell & Molec Immunol 7:260-2, 2012). Mast cells release leukotrienes, histamine,prostaglandins, and cytokines to increase vascular permeability andbronchoconstriction, and recruit other immune cells such as neutrophils,eosinophils and T lymphocytes to the site (Henderson et al., JEM184:1483-94, 1996; White et al., JACI 86:599-605, 1990). Additionally,they enhance immune responses by inducing adhesion molecule upregulationon endothelial cells to increase immune cell trafficking (Meng et al., JCell Physiol 165:40-53, 1995). Mast cells play an important role inairway remodeling; in asthmatics, an increased number of mast cells isfound within the airway smooth muscle (ASM) cell layer, and secretemediators to promote ASM proliferation (reviewed by Okayama et al., CurrOpin Immunol 19:687-93, 2007).

Inflammatory pulmonary condition is an example of an inflammatorycondition. Exemplary inflammatory pulmonary conditions includeinfection-induced pulmonary conditions including those associated withviral, bacterial, fungal, parasite or prion infections; allergen-inducedpulmonary conditions; pollutant-induced pulmonary conditions such asasbestosis, silicosis, or berylliosis; gastric aspiration-inducedpulmonary conditions, immune dysregulation, inflammatory conditions withgenetic predisposition such as cystic fibrosis, and physicaltrauma-induced pulmonary conditions, such as ventilator injury. Theseinflammatory conditions also include asthma, emphysema, bronchitis,chronic obstructive pulmonary disease (COPD), sarcoidosis,histiocytosis, lymphangiomyomatosis, acute lung injury, acuterespiratory distress syndrome, chronic lung disease, bronchopulmonarydysplasia, community-acquired pneumonia, nosocomial pneumonia,ventilator-associated pneumonia, sepsis, viral pneumonia, influenzainfection, parainfluenza infection, rotavirus infection, humanmetapneumovirus infection, respiratory syncitial virus infection andAspergillus or other fungal infections. Exemplary infection-associatedinflammatory diseases may include viral or bacterial pneumonia,including severe pneumonia, cystic fibrosis, bronchitis, airwayexacerbations and acute respiratory distress syndrome (ARDS). Suchinfection-associated conditions may involve multiple infections such asa primary viral infection and a secondary bacterial infection.Dysregulated ST2L signaling may play a role in the pathology ofpulmonary diseases such as asthma and Chronic Obstructive PulmonaryDisease (COPD) (reviewed in Alcorn et al., Annu Rev Physiol 72:495-516,2010). Commonly used animal models for asthma and airway inflammationinclude the ovalbumin challenge model, methacholine sensitization modelsand sensitization with Aspergillus fumigatus (Hessel et al., Eur JPharmacol 293:401-12, 1995). Inhibition of cytokine and chemokineproduction from cultured human bronchial epithelial cells, bronchialfibroblasts or airway smooth muscle cells can also be used as in vitromodels. The administration of antagonists of the present invention toany of these models can be used to evaluate the use of those antagoniststo ameliorate symptoms and alter the course of asthma, airwayinflammation, COPD and the like.

Asthma is an inflammatory disease of the lung that is characterized byairway hyperresponsiveness (“AHR”), bronchoconstriction, wheezing,eosinophilic or neutrophilic inflammation, mucus hypersecretion,subepithelial fibrosis, and elevated IgE levels. Patients with asthmaexperience “exacerbations”, a worsening of symptoms, most commonly dueto microbial infections of the respiratory tract (e.g. rhinovirus,influenza virus, Haemophilus influenza, etc.). Asthmatic attacks can betriggered by environmental factors (e.g. ascarids, insects, animals(e.g., cats, dogs, rabbits, mice, rats, hamsters, guinea pigs andbirds), fungi, air pollutants (e.g., tobacco smoke), irritant gases,fumes, vapors, aerosols, chemicals, pollen, exercise, or cold air. Apartfrom asthma, several chronic inflammatory diseases affecting the lungare characterized by neutrophil infiltration to the airways, for examplechronic obstructive pulmonary disease (COPD), bacterial pneumonia andcystic fibrosis (Linden et al., Eur Respir J 15:973-7, 2000; Rahman etal., Clin Immunol 115:268-76, 2005), and diseases such as COPD, allergicrhinitis, and cystic fibrosis are characterized by airwayhyperresponsiveness (Fahy and O'Byrne, Am J Respir Crit. Care Med163:822-3, 2001). Commonly used animal models for asthma and airwayinflammation include the model of methacholine challenge after ovalbuminsensitization and challenge (Hessel et al., Eur J Pharmacol 293:401-12,1995). Inhibition of cytokine and chemokine production from culturedhuman bronchial epithelial cells, bronchial fibroblasts or airway smoothmuscle cells can also be used as in vitro models. The administration ofantibody antagonists of the present invention to any of these models canbe used to evaluate the use of those antagonists to ameliorate symptomsand alter the course of asthma, airway inflammation, COPD and the like.

IL-33 signaling through the ST2L receptor on TH2 cells, basophils, mastcells, and the newly described Innate Lymphoid Type 2 Cells results inIL-5 and IL-13 (type 2 cytokine) secretion (ILCs reviewed by Spits etal., Nature Reviews Immunology 13:145-149, 2013). Beneficial effects oftherapeutics targeting IL-5 or IL-13 in asthma confirm the relevance ofthese pathways. IL-5 activates eosinophils, and treatment of a subgroupof severe asthmatics with sputum eosinophilia with a monoclonal antibodythat neutralizes IL-5 resulted in fewer exacerbations (Nair et al. NEngl J. Med. 2009; 360(10):985-93). IL-13 is reported to contribute toIgE synthesis, mucus secretion and fibrosis. Treatment of severeasthmatics with an anti-IL-13 monoclonal antibody resulted in animprovement in lung function, with a subgroup demonstrating a greaterimprovement (Corren et al., N. Engl. J. Med., 365:1088-1098, 2011).Other mediators of differential immunological pathways are also involvedin asthma pathogenesis, and blocking these mediators, in addition toST2L, may offer additional therapeutic benefit. Therapies that targetmultiple type 2 cytokines, or pathways upstream of type 2 cytokineproduction, could be beneficial in severe disease.

The VH and the VL domains of the ST2L antibodies of the invention may beincorporated into bispecific antibodies and molecules described herein,in which the bispecific antibody specifically binds Domain I of ST2L anda second antigen such as TSLP (thymic stromal lympohpoietin), IL-25,IL-17RB or TSLPR.

IL-25 and TSLP, like IL-33, trigger type 2 cytokine release via distinctsignaling complexes: IL-25 (IL-17E) is a member of the IL-17 family andsignals through IL-17RA/IL-17RB, and TSLP is a member of the IL-7 familyand signals through the TSLPR/IL-7Rα heterodimers (reviewed by Koyasu etal., Immunol 132:475-481, 2011). Animals deficient in IL-33, ST2L,IL-25, IL-17RB, TSLP, or TSLPR demonstrate less severe airwayinflammation in at least one of many different types of mouse models ofasthma; however lack of protection from airway inflammation may bepresent in most of these animal models, raising the possibility thatexposure of the epithelium to various allergens or pathogens couldtrigger release of IL-33, IL-25, and TSLP concomitantly. Hammad et al.reported that administration of house dust mite extract to mice resultedin the release of IL-25, TSLP and IL-33 (as well as IL-5 and IL-13downstream of IL-33) into the airway (Hammad et al., Nat Med 15:210-216,2009). This suggests that blocking ST2L and TSLP and/or IL-25 may havebeneficial effects, particularly in severe airway disease.

In another embodiment of the invention the antibody antagonistsspecifically binding Domain I of human ST2L can be used to generatebispecific molecules that bind ST2L and TSLP, ST2L and IL-25, ST2L andTSLPR, ST2L and IL-17RA, or ST2L and IL-17RB.

In another embodiment of the invention, the antibody antagonistsspecifically binding Domain I of human ST2L is a bispecific antibody,wherein the antibody further binds TSLP, IL-25, TSLPR, IL-17RA orIL-17RB.

TSLP, IL-25, TSLPR, IL-17RA and IL-17RB binding antibodies can begenerated using methods described herein, such as immunizing miceexpressing human immunoglobulin loci (Lonberg et al., Nature 368:856-9,1994; Fishwild et al., Nature Biotechnology 14:845-51, 1996; Mendez etal., Nature Genetics 15:146-56, 1997, U.S. Pat. Nos. 5,770,429,7,041,870, and 5,939,598) or Balb/c mice with the corresponding proteinsor extracellular domains of the proteins, or using phage displaylibraries as described herein. Alternatively, existing antibodies toTSLP, IL-25, TSLPR, IL-17RA and IL-17RB can be used to generate thebispecific molecules. Exemplary IL-25 antibodies that can be used arethose described in for example Int. Pat. Publ. No. WO2011/123507.

Arthritis, including osteoarthritis, rheumatoid arthritis, arthriticjoints as a result of injury, and the like, are common inflammatoryconditions, which would benefit from the therapeutic use ofanti-inflammatory proteins, such as the antagonists of the presentinvention. Activation of ST2L signaling may perpetuate inflammation andfurther tissue damage in the inflamed joint. Several animal models forrheumatoid arthritis are known. For example, in the collagen-inducedarthritis (CIA) model, mice develop chronic inflammatory arthritis thatclosely resembles human rheumatoid arthritis. ST2L-deficient (ST2KO)mice developed attenuated disease in this model, and pathology in thismodel was dependent on ST2L expression by mast cells (Xu et al., PNAS105:10913-8, 2008). In this model, there was reduced infiltration ofmononuclear and polymorphonuclear cells and of synovial hyperplasia inthe joints of ST2KO mice. The draining LNs of ST2KO mice cultured withcollagen (CII) showed significantly decreased IL-17, IFNγ, and TNFαproduction. ST2L-deficient mice adoptively transferred with wild-type(WT) bone marrow-derived mast cells (BMMC), before CIA was induced,developed more severe CIA than those engrafted with ST2KO BMMCs.Therefore ST2L signaling by mast cells was critical to the developmentof arthritis in a mouse model that resembles human rheumatoid arthritis.Administration of the ST2L antibodies of the present invention, whichinhibit mast cell responses, to the CIA model mice can be used toevaluate the use of these antagonists to ameliorate symptoms and alterthe course of disease.

Exemplary gastrointestinal inflammatory conditions are inflammatorybowel disease (IBD), ulcerative colitis (UC) and Crohn's disease (CD),colitis induced by environmental insults (e.g., gastrointestinalinflammation (e.g., colitis) caused by or associated with (e.g., as aside effect) a therapeutic regimen, such as administration ofchemotherapy, radiation therapy, and the like), infections colitis,ischemic colitis, collagenous or lymphocytic colitis, necrotizingenterocolitis, colitis in conditions such as chronic granulomatousdisease or celiac disease, food allergies, gastritis, infectiousgastritis or enterocolitis (e.g., Helicobacter pylori-infected chronicactive gastritis) and other forms of gastrointestinal inflammationcaused by an infectious agent. Several animal models forgastrointestinal inflammatory conditions exist. Some of the most widelyused models are the 2,4,6-trinitrobenesulfonic acid/ethanol(TNBS)-induced colitis model or the oxazalone model, which inducechronic inflammation and ulceration in the colon (Neurath et al., InternRev Immunol 19:51-62, 2000). Another model uses dextran sulfate sodium(DSS), which induces an acute colitis manifested by bloody diarrhea,weight loss, shortening of the colon and mucosal ulceration withneutrophil infiltration. Another model involves the adoptive transfer ofnaïve CD45RB^(high) CD4 T cells to RAG or SCID mice. In this model,donor naïve T cells attack the recipient gut causing chronic bowelinflammation and symptoms similar to human inflammatory bowel diseases(Read and Powrie, Curr Protoc Immunol Chapter 15 unit 15.13, 2001). Theadministration of antagonists of the present invention in any of thesemodels can be used to evaluate the potential efficacy of thoseantagonists to ameliorate symptoms and alter the course of diseasesassociated with inflammation in the gut, such as inflammatory boweldisease.

Renal fibrosis can develop from either an acute insult such as graftischemia/reperfusion (Freese et al., Nephrol Dial Transplant 16:2401-6,2001) or chronic condition such as diabetes (Ritz et al., Nephrol DialTransplant 11 Suppl 9:38-44, 1996). The pathogenesis is typicallycharacterized by an initial inflammatory response followed by sustainedfibrogenesis of the glomerular filtration apparatus and tubularinterstitium (Liu, Kidney Int 69:213-7, 2006). Tubulointerstitialfibrosis has been shown to play a critical role in the pathogenesis ofrenal injury to end-stage renal failure and the proximal tubule cell hasbeen revealed as a central mediator (Phillips and Steadman, HistolHistopathol 17:247-52, 2002; Phillips, Chang Gung Med J 30:2-6, 2007).Fibrogenesis in the tubulointerstitial compartment is mediated in partby activation of resident fibroblasts, which secrete pro-inflammatorycytokines that stimulate the proximal tubule epithelium to secrete localinflammatory and fibrogenic mediators. Additionally, chemotacticcytokines are secreted by fibroblasts and epithelial cells and provide adirectional gradient guiding the infiltration of monocytes/macrophagesand T-cells into the tubulointerstitium. The inflammatory infiltrateproduces additional fibrogenic and inflammatory cytokines that furtheractivate fibroblast and epithelial cytokine release while alsostimulating the epithelium to undergo a phenotypic transition in whichthe cells deposit excess extracellular matrix components (Simonson,Kidney Int 71:846-54, 2007).

Other exemplary fibrotic conditions may include liver fibrosis(including but not limited to alcohol-induced cirrhosis, viral-inducedcirrhosis, autoimmune-induced hepatitis); lung fibrosis (including butnot limited to scleroderma, idiopathic pulmonary fibrosis); kidneyfibrosis (including but not limited to scleroderma, diabetic nephritis,glomerular nephritis, lupus nephritis); dermal fibrosis (including butnot limited to scleroderma, hypertrophic and keloid scarring, burns);myelofibrosis; neurofibromatosis; fibroma; intestinal fibrosis; andfibrotic adhesions resulting from surgical procedures. The fibrosis canbe organ specific fibrosis or systemic fibrosis. The organ specificfibrosis can be associated with lung fibrosis, liver fibrosis, kidneyfibrosis, heart fibrosis, vascular fibrosis, skin fibrosis, eye fibrosisor bone marrow fibrosis. The lung fibrosis can be associated withidiopathic pulmonary fibrosis, drug induced pulmonary fibrosis, asthma,sarcoidosis or chronic obstructive pulmonary disease. The liver fibrosiscan be associated with cirrhosis, schistomasomiasis or cholangitis. Thecirrhosis can be selected from alcoholic cirrhosis, post-hepatitis Ccirrhosis, primary biliary cirrhosis. The cholangitis can be sclerosingcholangitis. The kidney fibrosis can be associated with diabeticnephropathy or lupus glomeruloschelerosis. The heart fibrosis can beassociated with myocardial infarction. The vascular fibrosis can beassociated with postangioplasty arterial restenosis or atherosclerosis.The skin fibrosis can be associated with burn scarring, hypertrophicscarring, keloid, or nephrogenic fibrosing dermatopathy. The eyefibrosis can be associated with retro-orbital fibrosis, postcataractsurgery or proliferative vitreoretinopathy. The bone marrow fibrosis canbe associated with idiopathic myelofibrosis or drug inducedmyelofibrosis. The systemic fibrosis can be systemic sclerosis or graftversus host disease.

Other inflammatory conditions and neuropathies, which may be preventedor treated by the methods of the invention are those caused byautoimmune diseases. These conditions and neuropathies include multiplesclerosis, systemic lupus erythematous, and neurodegenerative andcentral nervous system (CNS) disorders including Alzheimer's disease,Parkinson's disease, Huntington's disease, bipolar disorder andAmyotrophic Lateral Sclerosis (ALS), liver diseases including primarybiliary cirrhosis, primary sclerosing cholangitis, non-alcoholic fattyliver disease/steatohepatitis, fibrosis, hepatitis C virus (HCV) andhepatitis B virus (HBV), diabetes and insulin resistance, cardiovasculardisorders including atherosclerosis, cerebral hemorrhage, stroke andmyocardial infarction, arthritis, rheumatoid arthritis, psoriaticarthritis and juvenile rheumatoid arthritis (JRA), osteoporosis,osteoarthritis, pancreatitis, fibrosis, encephalitis, psoriasis, Giantcell arteritis, ankylosing spondolytis, autoimmune hepatitis, humanimmunodeficiency virus (HIV), inflammatory skin conditions, transplant,cancer, allergies, endocrine diseases, wound repair, other autoimmunedisorders, airway hyperresponsiveness and cell, virus, or prion-mediatedinfections or disorders.

One embodiment of the invention is method of treating or preventing aST2L-mediated condition comprising administering a therapeuticallyeffective amount of an isolated human or human-adapted antibodyantagonist that specifically binds Domain I (SEQ ID NO: 9) of humanST2L, blocks IL-33/ST2L interaction, competes for binding to human ST2L(SEQ ID NO: 1) with an isolated antibody comprising a heavy chainvariable region (VH) of SEQ ID NO: 47 and a light chain variable region(VL) of SEQ ID NO: 51 and/or binds human ST2L at amino acid residues35-48 of SEQ ID NO: 1 (RCPRQGKPSYTVDW; SEQ ID NO: 210) to a patient inneed thereof for a time sufficient to treat or prevent the ST2L-mediatedcondition.

Another embodiment of the invention is a method of inhibiting mast cellresponse in a patient comprising administering a therapeuticallyeffective amount of an isolated human or human-adapted antibodyantagonist that specifically binds Domain I (SEQ ID NO: 9) of humanST2L, blocks IL-33/ST2L interaction, competes for binding to human ST2L(SEQ ID NO: 1) with an isolated antibody comprising a heavy chainvariable region (VH) of SEQ ID NO: 47 and a light chain variable region(VL) of SEQ ID NO: 51 and/or binds human ST2L at amino acid residues35-48 of SEQ ID NO: 1 (RCPRQGKPSYTVDW; SEQ ID NO: 210) to a patient inneed thereof for a time sufficient to inhibit the mast cell response.

Another embodiment of the invention is a method of inhibitinginteraction of IL-33 and ST2L in a subject, comprising administering tothe subject an isolated human or human-adapted antibody antagonist thatspecifically binds Domain I (SEQ ID NO: 9) of human ST2L, blocksIL-33/ST2L interaction, competes for binding to human ST2L (SEQ IDNO: 1) with an isolated antibody comprising a heavy chain variableregion (VH) of SEQ ID NO: 47 and a light chain variable region (VL) ofSEQ ID NO: 51 and/or binds human ST2L at amino acid residues 35-48 ofSEQ ID NO: 1 (RCPRQGKPSYTVDW; SEQ ID NO: 210) in an amount sufficient toinhibit the interaction of IL-33 and ST2L.

In another embodiment, the ST2L-mediated condition is asthma, airwayhyper-reactivity, sarcoidosis, chronic obstructive pulmonary disease(COPD), idiopathic pulmonary fibrosis (IPF), cystic fibrosis,inflammatory bowel disease, (IBD), eosinophilic esophagitis,scleroderma, atopic dermatitis, allergic rhinitis, bullous pemphigoid,chronic urticaria, diabetic nephropathy, rheumatoid arthritis,interstitial cystitis or Graft Versus Host Disease (GVHD).

In another embodiment, the ST2L-mediated condition is associated withinflammatory cell recruitment in lung, goblet cell hyperplasia, orincreased mucous secretion.

In another embodiment, the ST2L-mediated condition is associated withmast cell response.

In another embodiment, the inhibiting mast cell response comprisesinhibiting the level of GM-CSF, IL-5, IL-8, IL-10 or IL-13 released byhuman cord blood-derived mast cells by at least 50% with 50 μg/mlantibody.

In another embodiment, the antibody antagonist administered to a patientin need thereof is a bispecific antibody that specifically binds DomainI (SEQ ID NO: 9) of human ST2L, blocks IL-33/ST2L interaction, competesfor binding to human ST2L (SEQ ID NO: 1) with an isolated antibodycomprising a heavy chain variable region (VH) of SEQ ID NO: 47 and alight chain variable region (VL) of SEQ ID NO: 51 and/or binds humanST2L at amino acid residues 35-48 of SEQ ID NO: 1 (RCPRQGKPSYTVDW; SEQID NO: 210), and further binds TSLP, IL-25, TSLPR, IL-17RA or IL-17RB.

Administration/Pharmaceutical Compositions

The “therapeutically effective amount” of the anti-ST2L antibodieseffective in the treatment of conditions where modulation of ST2Lbiological activity is desirable can be determined by standard researchtechniques. For example, the dosage of the anti-ST2L antibody that willbe effective in the treatment of an inflammatory condition such asasthma or rheumatoid arthritis can be determined by administering theanti-ST2L antibody to relevant animal models, such as the modelsdescribed herein.

In addition, in vitro assays can optionally be employed to help identifyoptimal dosage ranges. Selection of a particular effective dose can bedetermined (e.g., via clinical trials) by those skilled in the art basedupon the consideration of several factors. Such factors include thedisease to be treated or prevented, the symptoms involved, the patient'sbody mass, the patient's immune status and other factors known by theskilled artisan. The precise dose to be employed in the formulation willalso depend on the route of administration, and the severity of disease,and should be decided according to the judgment of the practitioner andeach patient's circumstances. Effective doses can be extrapolated fromdose-response curves derived from in vitro or animal model test systems.

The mode of administration for therapeutic use of the antibody of theinvention may be any suitable route that delivers the agent to the host.Pharmaceutical compositions of these antibodies are particularly usefulfor parenteral administration, e.g., intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, or intranasal.

The antibody of the invention may be prepared as pharmaceuticalcompositions containing an effective amount of the agent as an activeingredient in a pharmaceutically acceptable carrier. The term “carrier”refers to a diluent, adjuvant, excipient, or vehicle with which theactive compound is administered. Such pharmaceutical vehicles can beliquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil and the like. For example, 0.4% saline and 0.3% glycinecan be used. These solutions are sterile and generally free ofparticulate matter. They may be sterilized by conventional, well-knownsterilization techniques (e.g., filtration). The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, stabilizing, thickening, lubricating and coloring agents, etc.The concentration of the antibody of the invention in suchpharmaceutical formulation can vary widely, i.e., from less than about0.5%, usually at or at least about 1% to as much as 15 or 20% by weightand will be selected primarily based on required dose, fluid volumes,viscosities, etc., according to the particular mode of administrationselected.

Thus, a pharmaceutical composition of the invention for intramuscularinjection could be prepared to contain 1 ml sterile buffered water, andbetween about 1 ng to about 100 mg, e.g. about 50 ng to about 30 mg ormore preferably, about 5 mg to about 25 mg, of an anti-ST2L antibody ofthe invention. Similarly, a pharmaceutical composition of the inventionfor intravenous infusion could be made up to contain about 250 ml ofsterile Ringer's solution, and about 1 mg to about 30 mg and preferably5 mg to about 25 mg of an antagonist of the invention. Actual methodsfor preparing parenterally administrable compositions are well known andare described in more detail in, for example, “Remington'sPharmaceutical Science”, 15th ed., Mack Publishing Company, Easton, Pa.

The antibodies of the invention can be lyophilized for storage andreconstituted in a suitable carrier prior to use. This technique hasbeen shown to be effective with conventional immunoglobulins and proteinpreparations and art-known lyophilization and reconstitution techniquescan be employed.

The present invention will now be described with reference to thefollowing specific, non-limiting examples.

Materials And Methods (General)

Human and Cynomolgus (Macaca fascicularis, Cyno) Receptor-Ligand BindingInhibition Assay (RLB Assay)

96-well plate was coated with 50 μl of 4 μg/ml human ST2L-ECD (aminoacids 19-328 of SEQ ID NO: 1) or 2 μg/ml cyno ST2L-ECD (amino acids19-321 of SEQ ID NO: 2) having C-terminal His₆ tag in bicarbonate bufferat 4° C. for 16 hrs. All subsequent steps were performed at roomtemperature. Plate was blocked with 200 μl blocking buffer, and waswashed 3 times with 300 μl of wash buffer containing PBS+0.05% Tween. 30μl of various dilutions of anti-ST2L mAbs were added to the wells andincubated for 1 hour. For human receptor-ligand binding assay 20 μl ofbiotinylated human IL-33 (residues 112-270 of SEQ ID NO: 3) was added at100 ng/ml final concentration and incubated for 30 minutes. For cynoreceptor-ligand binding assay 20 μl of biotinylated cyno IL-33 (residues112-269 of SEQ ID NO: 4) was added at 200 ng/ml final concentration andincubated for 30 minutes. The plate was washed 3 times with 300 μl ofwash buffer. 50 μl of 0.2 μg/ml Streptavidin-HRP (JacksonImmunoresearch) was added and incubated for 30 min. The plate was washed3 times with 300 μl of wash buffer containing PBS+0.05% Tween. 50 μl ofTMB substrate (EMD Biosciences) was added to each well. Reaction wasstopped by the addition of 100 μl of 0.2N Sulfuric Acid. OD450 weremeasured using Envision plate reader (Perkin Elmer).

Generation of Chimeric ST2L Constructs

Various construct featuring human and mouse ST2L Domain I, II and IIIswaps were designed and generated using standard molecular biologytechniques. The constructs are listed in Table 1. Amino acid numberingcorresponds to human ST2L (hST2L) (SEQ ID NO: 1; NP_(—)057316) and mouseST2L (mST2L) (SEQ ID NO: 5; NP_(—)001020773) proteins.

TABLE 1 Origin of amino acid residues for each Construct Domain inchimeric constructs Name Domain I Domain II Domain III HHM-ST2L hST2Laa. 19-122 hST2L aa. 123-202 mST2L aa. 209-324 MHM-ST2L mST2L aa. 28-128hST2L aa. 123-202 mST2L aa. 209-324 HMH-ST2L hST2L aa. 19-122 mST2L aa.129-208 hST2L aa. 203-321 HH-ST2L hST2L aa.19-122 hST2L aa. 123-205 N/AhST2L: human ST2L SEQ ID NO: 1 mST2L: mouse ST2L SEQ ID NO: 5Domain Binding Determination Assay.

Antibody binding to ST2L domain I, II and III was determined usingstandard capture ELISA assay using electrochemiluminescent detectionformat (Meso-Scale Discovery (MSD) technology). 10 μg/mL of eachantibody was coated onto each well of an MSD HighBind plate (5 μL/well)for 2 hr at room temperature. The plate was blocked with 150 μL of 5%MSD Blocking buffer for 2 hr at room temperature, and washed 3 timeswith HEPES wash buffer, followed by the addition of 25 μL of sulfo taglabeled huST2L-ECD or mouse ST2L-ECD (amino acids 28-326 of SEQ ID NO:5) or HHM-ST2L (SEQ ID NO:6) or HMH-ST2L (SEQ ID NO: 8) chimeras orHH-ST2L (residues 19-205 of SEQ ID NO: 1) to the plate in increasingconcentrations from 5 nM to 40 nM. The plate was covered with aluminumfoil and incubated for 1 hr at room temperature with gentle shaking. Theplate was then washed 3 times with HEPES wash buffer. MSD read buffer(150 μl) was added to each well, and the plate was then read using anMSD Sector Imager 6000.

Those antibodies bound by human ST2L-ECD, HHM-ST2L and HMH-ST2L, but notby mouse ST2L-ECD recognize Domain I of human ST2L-ECD. Antibodies boundby human ST2L-ECD and HMH-ST2L, but not HHM-ST2L and mouse ST2L-ECD,recognize Domain III of human ST2L-ECD. Antibodies bound by human andmouse ST2L-ECD but not HH-ST2L recognize Domain III of human and mouseST2L-ECD.

Affinity Measurements of Anti-ST2L mAbs.

Anti-ST2L mAbs, huST2L-ECD and cynoST2L-ECD were expressed usingstandard methods. Goat anti-human IgG Fcγ fragment-specific Ab(cat#109-005-098) was obtained from Jackson ImmunoResearch laboratories(West Grove, Pa.). GLC sensor chips (Bio-Rad cat#176-5011), CM-5 sensorchips (GE Healthcare cat#BR100014) and reagents for preparation of thecapture surface were obtained from Biacore (GE healthcare, Piscataway,N.J.) or from Bio-Rad Life Sciences (Bio-Rad, Hercules, Calif.).

The interactions of anti-ST2L antibodies with His₆-tagged human ST2L-ECDand His₆-tagged cyno ST2L-ECD were studied by ProteOn using a ProteOnXPR36 at 25° C. A biosensor surface was prepared by coupling goatanti-human IgG Fcγ fragment specific antibody (Ab) to the surface of aGLC sensor chip using the manufacturer instructions for amine-couplingchemistry. The coupling buffer was 10 mM sodium acetate, pH 4.5. Thegoat anti-human IgG Fcγ (about 4500 response units) was immobilized inthe horizontal orientation. The anti-ST2L antibodies were providedpurified, or in crude supernatants. In either case these antibodies werediluted in PRB (PBS pH 7.4, supplemented with, 3 mM EDTA, and 0.005%Tween 20) to a concentration of about 0.5 μg/mL. The antibodies werecaptured (60-130 RU) in the vertical orientation onto the anti-human Fcγantibody-modified GLC chip. Capture of anti-ST2L mAbs was followed byinjection of huST2L ECD in solution (0.024 to 15 nM in 5-fold dilutions)or cynoST2L ECD in solution (0.020-5 nM in 4-fold dilutions) in thehorizontal orientation. The association was monitored for 4 minutes inall experiments (200 μL injected at 50 μL/min). The dissociation wasmonitored for 30 minutes. Regeneration of the sensor surface wasobtained with three 15 sec pulses of 10 mM glycine pH 1.5. The data werefit using the ProteOn software and using a 1:1 binding model with masstransfer.

Biacore experiments were performed using a Biacore 2000 or a Biacore3000 optical biosensor (Biacore AB). All experiments were run in BRB(PBS pH 7.4, supplemented with 3 mM EDTA and 0.005% Tween 20) with orwithout 0.1% BSA at 25° C.

A Biacore biosensor surface was prepared by coupling goat anti-human IgGFcγ fragment specific Ab to the carboxymethylated dextran surface of aCM-5 chip using manufacturer instructions for amine-coupling chemistry.The coupling buffer was 10 mM sodium acetate, pH 4.5. An average of 6000response units (RU) of Ab were immobilized in each of four flow cells.The anti-ST2L mAbs were captured (about 33 RU) onto the anti-human Fcγantibody-modified sensor chip surface. Capture of anti-ST2L mAbs wasfollowed by injection of huST2L ECD in solution (0.2 to 15 nM in 3-folddilutions) or cynoST2L ECD in solution (0.2 to 15 nM or 0.020-5 nM, in3-fold dilutions). The association was monitored for 4 minutes or 8minutes (200 μL injected at 50 μl/min or 20 μL/min for C2521 and C2519).The dissociation was monitored for 10 minutes, or up to 2.5 hours.Regeneration of the sensor surface was obtained with injection of 50 mMNaOH and/or injection of 100 mM H₃PO₄.

Data were processed using the Scrubber software, version 1.1 g (BiologicSoftware). Double reference subtraction of the data was performed bysubtracting the curves generated by buffer injection from thereference-subtracted curves for analyte injections to correct for buffercontribution to the signal and instrument noise (Myszka, Journal of MolRecogn 12:279-84, 1999).

After data processing, the data generated for kinetic and affinitydetermination were analyzed using the Scrubber software or theBIAevaluation software, version 4.0.1 (Biacore, AB). The kinetic datawere analyzed using a simple 1:1 binding model including a term for masstransfer.

Affinity Measurement of Anti-Mouse ST2L mAb (C1999/CNTO3914) to MurineST2L ECD.

Anti-ST2L mAb (C1999/CNTO3914) and murine ST2L extracellular domain(muST2L-ECD) were expressed and purified using standard methods.Anti-murine IgG Fcγ fragment-specific Ab was obtained from JacksonImmunoResearch laboratories (West Grove, Pa.). Sensor chips and reagentsfor preparation of the capture surface were obtained from Biacore (GEhealthcare, Piscataway, N.J.). The experimental Biacore running buffer(BRB) contained PBS pH 7.4 with 0.005% Tween 20 and 0.1 mg/mL BSA anddata were collected at 25° C.

The interaction of anti-ST2L antibody with muST2L-ECD was studied on aBiacore2000 at 25° C. A biosensor surface was prepared by couplinganti-mouse-Fc specific antibody to the surface of a CM4 sensor chipusing the manufacturer instructions for amine-coupling chemistry.C1999/CNTO3914 and muST2L-ECD were diluted in BRB. C1999 was capturedusing the anti-mouse Fcγ antibody (about 85 RU). Capture was followed byinjection muST2L ECD (residues 28-326 of SEQ ID NO: 5) in solution(starting at 15 nM, 5 concentrations, in a 3-fold serial dilution). Theassociation was monitored for 8 minutes. The dissociation was monitoredfor up 6000 seconds. The regenerations were performed using a 1/100dilution of phosphoric acid. The data were fit using a 1:1 bindingmodel.

Human Basophil Cell Line Assay (Basophil Cytokine Release Assay)

KU812 cells (human basophil cell line; ATCC, CRL-2099) were plated insterile 96-well U-bottom tissue culture plates at 25,000 or 50,000 cellsper well in a total 40 μl of RPMI 1640 growth medium (Invitrogen)supplemented with 10% FBS and penicillin/streptomycin. Anti-human ST2LmAbs and controls were added at various concentrations (50 μl/well) andincubated at 37° C. After 1 hour of incubation, recombinant “mature”IL-33 (amino acids 111-270 of SEQ ID NO: 3) was added at a finalconcentration of 10 ng/ml in 10 μl of RPMI growth medium. The cells werethen incubated at 37° C. for 18-24 hours to allow for IL-33-mediatedinduction of IL-5 and IL-6. Following incubation, the cells wereharvested and the cell supernatant was collected for subsequentdetection of IL-33-induced IL-5 and IL-6 using either ELISA (R&Dsystems) or bead-based multiplex analyses (Millipore).

Human Mast Cell Cytokine Release Assay and PGD₂ Release Assay

Mast cells were derived from CD34⁺ human cord blood cells (Lonza).Frozen vials of >1.0×10⁶ CD34⁺ cord blood cells were rapidly thawed andtransferred to a 50 ml conical tube. Drops of warmed or room tempStem-Pro 34 media+supplements (25 mls total; Invitrogen) were slowlyadded to the cells. The cells were centrifuged at 1,000 rpm for 15minutes and resuspended in media (10 mls of StemPro-34, with thefollowing supplements: 30 ng/ml IL-3, 100 ng/ml IL-6, and 100 ng/ml SCF.Cells were plated in 2 wells of a 6-well plate, and cultured for 1 week.On day 4, cells were expanded 1:3 in supplemented Stem Pro-34 media. Onday 7, non-adherent cells were removed and plated at 0.5×10⁶/ml inStemPro-34 media containing 10 ng/ml IL-6 and 100 ng/ml SCF. Cells wereexpanded weekly to maintain cell density at 0.5×10⁶/ml until mast cellswere mature at 6-10 weeks (assessed by expression of FcεR1, cKit, andtryptase).

Mature mast cells were cultured at 0.5×10⁶/ml in StemPro-34 andstimulated daily for 4 days in IL-4 (10 ng/ml; Peprotech), IL-6 (10ng/ml; R&D Systems) and SCF (100 ng/ml; Invitrogen). Prior to assay,cells were harvested, centrifuged at 1,000 RPM for 10 min andresuspended in fresh StemPro-34 media or RPMI containing 10% FCS withoutantibiotics, with 100 ng/ml human recombinant SCF. Cells were plated ata density of 65,000 to 75,000 cells/0.16 mls/well in a flat bottom,tissue culture-treated 96-well plate. The anti-ST2L mAbs were added tothe plate for a final concentration of 50, 10, 2, 0.4, 0.08, 0.016,0.0032 μg/ml for 30 minutes prior to the addition of IL-33. Recombinanthuman “mature” IL-33 (residues 111-270 of SEQ ID NO: 3) was alsoprepared at 10× (10 or 30 ng/ml) in media+100 ng/ml SCF. 20 μl of the10× IL-33 was added to the wells, for a final concentration of 1 (FIGS.6 and 7) or 3 ng/ml (FIG. 8), and the plates were incubated overnight at37° C., 5% CO₂. Culture supernatant was harvested 18-24 hours afterstimulation. The plates were centrifuged at 1,000 RPM for 10 minutes.The supernatant was removed and placed into a U bottom 96 well plate andstored at −20° C. prior to assaying. Human cytokine kits from Milliporewere used to analyze cytokine levels using Luminex™ technology. Levelsof PGD2 were measured using the Prostaglandin D2-MOX EIA kit from CaymanChemical Company, according to manufacturer's instructions. In order toenhance the sensitivity of the ELISA, PGD₂ in the mast cell culturesupernatants were converted into non-degradable MOX-PGD₂(methoxylsamine-PGD₂) by treatment with methoxylsamine hydrochloride(MOX-HCl).

Mouse Receptor-Ligand Binding Inhibition Assay (Mouse RLB Assay)

A 96-well clear plate (VWR) was coated with 50 μl of 2 μg/ml goatanti-human IgG, Fcγ fragment-specific (Jackson Immunoresearch) antibodyfor approximately 16 hours at 4° C. The remaining steps were completedat room temperature. Wells were incubated with blocking buffer, washedand 50 μl of 2 μg/ml mouse ST2L-ECD fused to human Fc was added for 1hour. The plate was washed and 1 μg/ml of biotinylated mIL-33 with orwithout anti-mST2L antibodies added. The plate was washed and detectiondone with streptavidin-HRP (Jackson Immune Research) and signaldeveloped with TMB substrate (RDI Division of Fitzgerald Industries)following manufacturer's instructions.

Mouse and Human Reporter Gene Assays (Human or Mouse RGA Assay)

HEK293 cells were plated at 50,000 cells per well in white clear-bottomtissue culture-treated 96-well plates (NUNC) in DMEM, 10% FBS andincubated in humidified incubator at 37° C., 5% CO₂ for 24 hours. Cellswere co-transfected with vectors encoding either human or mouse ST2L-ECDcDNA, NF-κB-Luciferase vector (Stratagene, Agilent Technologies, SantaClara, Calif.) using Lipofectamine™ 2000 in Opti-MEM media (Invitrogen)using standard protocols. After 24 hour incubation at 37° C., 5% CO₂,the transfected ells were treated with mouse (R&D Systems, residues109-266 of SEQ ID NO: 5) or human IL-33 (residues 112-270 or SEQ ID NO:3) with or without anti-ST2L antibodies for 16 hours at 37° C., 5% CO₂.Luciferase activity was measured using Steady-Glo® reagent (Promega)according to the manufacturer's instructions.

Mouse T-Cell Proliferation Assay

Mouse Th2 cells (D10.G4.1, ATCC) were cultured in complete growthmedium: RPMI 1640 medium with 2 mM L-glutamine adjusted to contain 1.5g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, and 1.0 mM sodiumpyruvate, and supplemented with: 0.05 mM 2-mercaptoethanol, 10 pg/mlIL-1α (R&D Systems), 10% fetal bovine serum, 10% rat T-STIM factor withCon A (rat IL-2 culture supplement available from Becton Dickinson). Thecells were washed twice with assay media (RPMI, 10% FBS, no IL-1, noT-STIM), resuspended at 1.25×10⁵ cells per ml and plated in 80 μl ofmedium in white clear bottom tissue culture treated 96-well plates(NUNC, Rochester, N.Y.). Various amounts of mouse IL-33 (residues109-266 of SEQ ID NO: 5) were added to the cells for the final assayvolume of 100 μl. When testing antibody neutralization, controlantibodies (spiked in spent hybridoma medium) or hybridoma supernatantswere added to the cells and incubated for 1 hour followed by addition of20 pg/ml mIL-33. The plates were cultured for 24 hours in humidifiedincubator at 37 C, 5% CO₂. Quantitation of viable cells was achievedwith CellTiter-Glo® reagents (Promega, Madison, Wis.); protocolperformed according to the manufacturer's instructions.

Mouse Bone Marrow Derived Mast Cell Assay

Mouse mast cells were derived from bone marrow of Balb/c mice (6 weeks).Cells were plated at 300,000 cells/well in RPMI media (endotoxin free),10% FBS, 10% WEHI cell line-conditioned medium, 10 ng/ml IL-3(Peprotech), 0.1 mM essential amino acids, 1% Penicillin/Streptomycin(Invitrogen). Anti-ST2L mAbs (100, 10, 1, 0.1, or 0.01 μg/ml) wereincubated with the cells for 1 hour prior to addition of recombinantmouse “mature” IL-33 (residues 109-266 of SEQ ID NO: 5 (10 ng/ml; R&DSystems). After approximately 24 h the supernatants were harvested andfrozen until analysis using the Millipore Mouse 22-plex kit forLuminex™, according to manufacturer's instructions.

Cyno Endothelial Cell Assay

Cynomolgus Aortic Endothelial cells cultured in EGM®-2 Endothelial CellGrowth Medium-2 (Lonza) were plated in 96-well tissue culture plates at10,000 or 20,000 cells per well. 50 μl of anti-ST2L antibodies wereadded to the cells starting at 100 μg/ml with subsequent 4- or 5-folddilutions and incubated at 37° C. for 1 hour before the addition ofrecombinant cyno ‘mature’ IL-33 (SEQ ID NO: 4). Fifty microliters of 20ng/ml cynomolgus IL-33 was then added to the cells and incubated at 37°C. for 24 hours. To evaluate IL-33-induced cytokine responses thesupernatants were harvested and the cytokine levels were assessed by aNon-Human Primate IL-8 kit for Luminex™ (Millipore) according tomanufacturer's instructions.

Mouse Peritoneal Lavage Assay

The peritoneums of 6 Balb/c mice was washed with a total of 3 ml PBS tocollect peritoneal cells. The majority of these cells were found to belymphocytes and macrophages, as determined by B220 and F4/80 expression(FACS analysis). Approximately 1% were cKit⁺ (CD117⁺) mast cells. Cellswere centrifuged and the pellet was resuspended to 1×10⁶ cells/ml inAlpha MEM media+10% FBS+100 U/ml Penicillin+100 μg/ml Streptomycin(Invitrogen). Cells were plated at 200 μl per well in a 96-well plateand rested 2 h at 37° C. Anti-ST2L mabs were added to the cells for 30minutes prior to the addition of 10 ng/ml mouse “mature” IL-33 (R&DSystems; residues 109-266 of SEQ ID NO: 215). Supernatants werecollected 24 h after IL-33 addition, stored at −20° C. until analysis,and analyzed using the Millipore Mouse 22-plex kit for Luminex™according to manufacturer's instructions.

Example 1 Generation of Rat Anti-Mouse ST2L Antibodies

Rats were immunized intraperitoneally with mouse ST2-Fc (R&D Systems(Ser27-Ser342 of SEQ ID NO: 5) and assessed for specific IgG titers.Once sufficient titers were obtained, splenocytes were isolated andfused with FO cells. The resulting hybridomas were plated in 96 wellplates or methylcellulose and cultured for 10 days. Antigen specificclones were identified by standard capture ELISA for binding to mST2-Fcand cross screened against the Fc protein alone. Murine ST2-specifichybridomas were further tested for the inhibition of IL-33 binding toST2 in an ELISA and for the inhibition of IL-33-induced D10.G4.1 mouseTh2 cell proliferation. Hybridomas exhibiting neutralization in bothreceptor-ligand binding and cell-based proliferation assays wereclonally selected by limiting dilution. Hybridoma V-regions weresequenced and cloned into mouse IgG1 background. ST2L-ECD domainspecificity was addressed by standard immunosorbent assay withelectrochemiluminescent detection using various human-mouse domain-swapconstructs.

Antibody secreted by hybridoma C1999 was cloned into mouse IgG1background and named CNTO3914. Sequences of CNTO3914 variable regionsand CDRs are shown in Table 2. CNTO3914 does not cross-react with humanST2L and binds Domain I of mouse ST2L-ECD.

TABLE 2   HCDR1 HCDR2 HCDR3 SEQ ID SEQ ID SEQ ID mAb Name Sequence NO:Sequence NO: Sequence NO: C1999/ HYGMA 13 SIITDGTSTYYRDSVKG 14 QSDDYFDY15 CNTO3914 LCDR1 LCDR2 LCDR3 SEQ ID SEQ ID SEQ ID mAb Name Sequence NO:Sequence NO: Sequence NO: C1999/ KSSQSLEYSDGDSYLE 16 GVSNRFS 17FQATHDPFT 18 CNTO3914 mAb Name SEQ ID NO: C1999/ VH sequence 19 CNTO3914EVQLVESGGGLLQPGRSLKLSCTASGFIFSHYGMAWVRQAPTKGLEWVSSIITDGTSTYYRDSVKGRFTISRDNAKNTQYLQMDSLRSEDTATYYCAR QSDDYFDYWGQGVMVTVSSVL sequence DVVLTQTPVSLSVTLGDQASISCKSSQSLEYSDGDSYLEWYLQKPGQSP 20QLLIYGVSNRFSGVPDRFIGSGSGTDFTLKISRVEPEDLGVYYCFQATHDP  FTFGSGTKLEIK

Example 2 Generation of Mouse Anti-Human ST2L Antibodies

Two different immunizations were performed for generation of mouseanti-human ST2 mAbs.

BALB/c were immunized intraperitoneally with soluble ST2-Fc (R&DSystems, SEQ ID NO: 157) and assessed for specific IgG titers. Oncesufficient titers were obtained, splenocytes were isolated and fusedwith FO cells. The resulting hybridomas were plated in 96 well platesand cultured for 10 days. Antigen specific clones were identified bystandard capture ELISA for binding to C-terminal His₆-tagged huST2L-ECDand cross-reactivity to His₆-tagged cyno ST2L-ECD. Human ST2L-specifichybridomas cross-reacting with cyno ST2L were further tested for theinhibition of IL-33 binding to huST2L in an ELISA assay and for theinhibition of NF-κB activation in reporter gene assay. Clones inhibitingin reporter gene assay or in both ELISA and reporter gene assay wereselected for further studies.

Antibodies from hybridomas C2494, C2519A and C2521A were selected forfurther analyses. C2519A and C2521A bind human ST2L at Domain III, andC2494 binds human ST2L at Domain I. Antibody C2494 was cloned into humanIgG2 background, and the full length antibody was named STLM62.

Anti-human ST2L mAbs were generated at Genovac Gmbh by proprietary DNAimmunization technology using full length ST2L constructs and boostingwith the cells transfected to express human ST2L-ECD. Hybridomas werescreened for binding to human ST2L-ECD by flow cytometry. Clones thatexhibited binding in this assay were confirmed to bind hST2L-ECD andfurther characterized for binding to cyno ST2L-ECD by standard captureELISA. Select clones were characterized in receptor-ligand bindinginhibition ELISA and reporter gene assay. Clones inhibiting in reportergene assay or in both ELISA and reporter gene assay were selected forfurther studies.

Antibody from Genovac hybridoma C2244 was selected for further analysesand cloned into human IgG2 background. The full length antibody wasnamed STLM15. STLM15 binds human ST2L at Domain I.

Sequences of the VH, VL and CDR domains of the mouse anti-humanantibodies are shown in Table 3.

TABLE 3 HCDR1 HCDR2 HCDR3 mAb SEQ ID SEQ ID SEQ ID Name Sequence NO:Sequence NO: Sequence NO: C2519A DYNMN 21 NINPYYGSTTYNQKFKG 25EGDTYLAWFAY 29 C2521A TYWMN 22 QIFPASGSTYYNEMFKD 26 SENIYYINFQYYFAY 30C2244/ SDYAWN 23 FISYSGDTSFNPSLKS 27 YDGYSFDY 31 STLM15 C2494/ DDYMH 24RIDPAIGNTEYAPKFQD 28 GDFYAMDY 32 STLM62 LCDR1 LCDR2 LCDR3 mAb SEQ IDSEQ ID SEQ ID Name Sequence NO: Sequence NO: Sequence NO: C2519ARSSQSIVYSNGNTYLE 33 KVSNRFS 37 FQGSHVPPT 41 C2521A RASQNIGTRMH 34YASESIS 38 QQSNTWPFT 42 C2244/ RASKSVSTSGSSYMF 35 LASNLES 39 QHSREIPYT43 STLM15 C2494/ ITNTDIDDVIH 36 EGNTLRP 40 LQSDNMLT 44 STLM62 mAbVH sequence SEQ ID NO: C2519AEFQLQQSGPELVKPGASVKISCKASGYSFTDYNMNWVKQSHGKSLEWI 45GNINPYYGSTTYNQKFKGKATLTVDKSSNTAYMHLNSLTSEDSAVYYCAREGDTYLAWFAYWGQGTLVTVSA C2521AQIQLQQSGPELVRPGTSVKISCKASGYTFLTYWMNWVKQRPGQGLEWI 46GQIFPASGSTYYNEMFKDKATLTVDTSSSTAYMQLSSLTSEDTAVYFCARSENIYYINFQYYFAYWGQGTTLTVSS C2244/EVQLQESGPGLVKPSQSLSLTCTVTGFSITSDYAWNWIRQFPGSKLEW 47 STLM15MGFISYSGDTSFNPSLKSRISVTRDTSKNQFFLQLNSVTTEDTATYYCASY DGYSFDYWGQGTTLTVSSC2494/ EVQLQQSVAELVRPGASVKLSCTASAFNIKDDYMHWVKQRPEQGLEW 48 STLM62IGRIDPAIGNTEYAPKFQDKATMTADTSSNTAYLQLSSLTSEDTAVYYCA LGDFYAMDYWGQGTSVTVSSmAb VL sequence SEQ ID NO: C2519ADVLMTQTPLSLPVSLGDQASISCRSSQSIVYSNGNTYLEWYLQKPGQSP 49KLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVP PTFGGGTKLEIK C2521AILLTQSPAILSVSPGERVSFSCRASQNIGTRMHWYQQRTNGSPRLLIKYA 50SESISGIPSRFSGSGSGTDFTLTISSVESEDIADYYCQQSNTWPFTFGSGTK LEIK C2244/DIVLTQSPASLAISLGQRATISCRASKSVSTSGSSYMFWYQQKPGQPPKL 51 STLM15LIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAAAYYCQHSREIPYT FGGGTKLEIK C2494/ETTVTQSPASLSVATGEKVTIRCITNTDIDDVIHWYQQKPGEPPKLLISEG 52 STLM62NTLRPGVPSRFSSSGYGTDFVFTIENTLSEDVADYYCLQSDNMLTFGAGT KLELK

Example 3 Generation of Fully Human ST2L Antibodies

Human ST2L-binding Fabs were selected from de novo pIX phage displaylibraries as described in Shi et al., J Mol Biol 397:385-96, 2010; Int.Pat. Publ. No. WO2009/085462; U.S. Pat. Publ. No. US2010/0021477).Briefly, the libraries were generated by diversifying human scaffoldswhere germline VH genes IGHV1-69*01, IGHV3-23*01, and IGHV5-51*01 wererecombined with the human IGHJ-4 minigene via the H3 loop, and humangermline VLkappa genes 012 (IGKV1-39*01), L6 (IGKV3-11*01), A27(IGKV3-20*01), and B3 (IGKV4-1*01) were recombined with the IGKJ-1minigene to assemble complete VH and VL domains. The positions in theheavy and light chain variable regions around H1, H2, L1, L2 and L3loops corresponding to positions identified to be frequently in contactwith protein and peptide antigens were chosen for diversification.Sequence diversity at selected positions was limited to residuesoccurring at each position in the IGHV or IGLV germline gene families ofthe respective IGHV or IGLV genes. Diversity at the H3 loop wasgenerated by utilizing short to mid-sized synthetic loops of lengths7-14 amino acids. The amino acid distribution at H3 was designed tomimic the observed variation of amino acids in human antibodies. Librarydesign is detailed in Shi et al., J Mol Biol 397:385-96, 2010. Thescaffolds utilized to generate libraries were named according to theirhuman VH and VL germline gene origin. The three heavy chain librarieswere combined with the four germline light chains or germline lightchain libraries to generate 24 unique VH:VL combinations for screening.All 24 VH:VL library combinations were utilized in phage panningexperiments against huST2L-ECD-Fc.

The libraries were panned using a Fc fusion of the huST2L-ECD (residues19-328 of SEQ ID NO: 1). Pannings were done in 2 different formats,antigen (Ag) in solution and Ag displayed. For Ag in solution thestreptavidin-coated magnetic beads were blocked in PBS with 3% non-fatdry milk. The biotinylated (Bt) antigen huST2L-ECD human Fc fusion(Bt-huST2L-ECD-Fc) with a 10× higher concentration of human Fc proteinas competitor was mixed with Fab-pIX phage libraries. The Fab-pIX phagebound to the Bt-huST2L-ECD-Fc was captured on the blocked streptavidin(SA)-coated magnetic beads. Phage selections were performed for threerounds where the huST2L-ECD-Fc concentrations varied from 100 nM, 10 nM,10 nM from rounds 1 to 3, respectively. For Ag display theBt-huST2L-ECD-Fc was coated on SA coated magnetic beads. Fab-pIX phagelibraries plus 10× excess of human Fc protein was added simultaneouslyto the Bt-huST2L-ECD-Fc displayed SA-magnetic beads. Bt-Agconcentrations used were 100 nM, 10 nM, 10 nM for rounds 1 to 3,respectively. Screening was done for both panning formats by ELISA forFab binding to huST2L-ECD-Fc protein. A total of 79 Fabs with binding tohST2L-Fc were isolated from these selections. Fab HuT2SU-39 wasdetermined by a ranking ELISA to have the best binding activity overall.

An ELISA based IL-33 binding inhibition assay was performed on the 79Fabs. A total of 32 Fabs showed inhibition of IL-33 binding tohuST2L-ECD-Fc. 46 Fabs were chosen for affinity maturation from the pIXde novo campaign.

Example 4 Affinity-Maturation of Fully Human ST2L Antibodies

Select antibodies were affinity-matured using an “in-line” maturationprocess described in Shi et al., J Mol Biol 397:385-96, 2010 andWO09085462A1. In this technology, the VH regions of Fab clones obtainedin the first selection are combined with libraries of the correspondingVL scaffold. All VH genes from the 46 Fabs identified in Example 3 werecloned into the appropriate VL maturation libraries as pools accordingto their original VL gene family. The used VL scaffold libraries andtheir diversification schemes are shown in Table 4. The human VLscaffolds are as follows: IGKV1-39*01 (O12), IGKV3-11 (L6), IGKV3-20(A27), IGKV4-1*01 (B3) and are described for example in U.S. Pat. Publ.No. US2012/0108795. For affinity maturation panning, the phage librarieswere added to Bt-huST2-ECD-Fc first. After incubation the maturationlibrary phage/Bt-hST2L-ECD-Fc complex was added to the SA-coatedmagnetic beads. The Bt-huST2-Fc concentrations varied respectively fromR1 to R3 at 10 nM, 1 nM, and 0.1 nM. The final wash of round 3 wasperformed overnight at room temperature in the presence of 10 nMunlabelled huST2L-ECD-Fc to further drive affinity improvement.

TABLE 4   VL library diversification scheme for different scaffoldsPosition Loop (Kabat) A27 B3 L6 O12 L1 30 SRNTD RNDGHSY SRNAD SRNAD 30aSNR RNDGHWY — 30e RNDGHSY — 31 SNRADH RNDGHWY NSKD SNKDG 32 YFHQSEK YNWRYWDFHSAN YHNDWFSAV L2 50 ADGS YWNK ADKGYFTN FYTNKADG L3 91 YSHA SYWHRYSGF SAYHPD 92 YNDSHIFKG SYGN RHNSL FIYHNDKGRE 93 SNTDGHR STER NDKRSTHNDRG 94 TYLVFAS WYSH WA TYLVFSRGPI 96 WYFLIR YRWH WYFLIR LWRFYIN

A total of 161 sequence unique Fabs were obtained from the maturationpannings. Fabs showing highest binding to huST2L-ECD were converted toIgG for further characterization.

MAbs ST2M48, ST2M49, ST2M50 and ST2M51 were selected for furthercharacterization, and their VH, VL and CDR sequences are shown in Table5. Mabs ST2M48, ST2M49, ST2M50 and ST2M51 bind human ST2L at Domain III,and cross-react with mouse ST2L.

TABLE 5 HCDR1 HCDR2 HCDR3 SEQ ID SEQ ID SEQ ID mAb ID HC ID Sequence NO:Sequence NO: Sequence NO: ST2M48 STLH125 TSYWIG 53 GIIYPGDSYTRYSPSFQG 55LSGRFDY 57 ST2M49 STLH149 TSYWIG 53 GIIYPGDSYTRYSPSFQG 55 IGGMFDY 58ST2M50 STLH125 TSYWIG 53 GIIYPGDSYTRYSPSFQG 55 LSGRFDY 57 ST2M51 STLH130SSYAIS 54 GIIPIFGTANYAQKFQG 56 DTPQLDY 59 LCDR1 LCDR2 LCDR3 SEQ IDSEQ ID SEQ ID mAb ID LC ID Sequence NO: Sequence NO: Sequence NO: ST2M48STLL232 RASQSVRDALA 60 FASNRAT 64 QQFNTWPIT 67 ST2M49 STLL216RASQSVANALA 61 KASNRAT 65 QQYYGWPIT 68 ST2M50 STLL228 RASQSVSNALA 62FASNRAT 64 QQFFNWPIT 69 ST2M51 TC1L3 RASQSISSYLN 63 YASSLQS 66 QQSYSTPLT70 SEQ ID mAb Name VH sequence NO: ST2M48EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWM 71GIIYPGDSYTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARLS GRFDYWGQGTLVTVSSST2M49 EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWM 72GIIYPGDSYTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARIG GMFDYWGQGTLVTVSSST2M50 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEW 71MGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR YNFFFDYWGQGTLVTVSSST2M51 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEW 73MGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR DTPQLDYWGQGTLVTVSSSEQ ID mAb Name VL sequence NO: ST2M48EIVLTQSPATLSLSPGERATLSCRASQSVRDALAWYQQKPGQAPRLLIYFA 74SNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFNTWPITFGQGT KVEIK ST2M49EIVLTQSPATLSLSPGERATLSCRASQSVANALAWYQQKPGQAPRLLIYKA 75SNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYYGWPITFGQGT KVEIK ST2M50EIVLTQSPATLSLSPGERATLSCRASQSVDDWLAWYQQKPGQAPRLLIYK 76ASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYNRAPWTFGQ GTKVEIK ST2M51DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYYA 77SSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTK VEIK

Example 5 Characterization of Anti-ST2L Antibodies

Antibodies derived from various campaigns as described above werefurther characterized for their ability to block IL-33/ST2L interaction,for their inhibition of IL-33-induced signaling as measured by NF-κBreporter gene assay, ability of the antibodies to inhibit mast cellresponses, for their affinity against human and cyno ST2L, andcross-reactivity with mouse ST2L. Epitope mapping was done usinghuman/mouse ST2L domain swap chimeric constructs as described inMaterials and Methods. Results of the experiments are shown in Tables 6,7 and 8. In Tables 7 and 8, “+” indicates that the antibody blocksIL-33/ST2L interaction, and “−” indicates it does not block IL-33/ST2Linteraction. Experiments with CNTO3914 were done using mouse cells andreagents due to lack of cross-reactivity to human. Human cells and humanreagents were used in assays for all other antibodies.

Characterized antibodies were grouped to those that block IL-33/ST2Linteraction (mAbs STLM15, STLM62 and CNTO3914) and those that do notblock the IL-33/ST2L interaction (mAbs C2519, C2521, ST2M48, ST2M49,ST2M50 and ST2M51). The antibodies blocking IL-33/ST2L interaction bindto ST2L Domain I, whereas the non-blocking antibodies bind to ST2LDomain III. The antibodies tested inhibited ST2L downstream signaling asassessed by the NF-κB reporter gene assay and IL-33-induced cytokinerelease by the KU812 human basophil cell line, or in case of CNTO3914,assessed by mouse Th2 cell proliferation. Antibodies binding to ST2LDomain I inhibited at higher level human mast cell responses as assessedby cytokine and chemokine secretion when compared to anti-ST2Lantibodies binding ST2L Domain III. CNTO3914, which binds mouse ST2Ldomain I and does not cross-react with human was also able to inhibitIL-33-induced mouse mast cell responses.

TABLE 6 Affinity to human ST2L Affinity to cyno ST2L corresponding K_(D)K_(D) mAb hybridoma k_(on) (M⁻¹s⁻¹) k_(off) (s⁻¹) (pM) k_(on) (M⁻¹s⁻¹)k_(off) (s⁻¹) (pM) STLM15 C2244 1.02E+06 4.25E−05 42 4.81E+06 5.30E−0511 STLM62 C2494 4.26E+06 1.19E−04 28 4.51E+07 5.39E−04 12 na C25194.83E+05 8.70E−05 180 7.14E+04 3.20E−03 44800 na C2521 6.18E+05 4.90E−0579 4.47E+05 1.66E−03 3710 ST2M48 na 1.32E+06 7.33E−05 56 1.03E+072.65E−03 257 ST2M49 na 1.59E+06 1.61E−04 101 4.66E+07 1.24E−02 266ST2M50 na 1.15E+06 5.10E−05 45 2.01E+07 2.49E−03 124 ST2M51 na 1.29E+064.87E−05 38 4.42E+07 3.36E−03 76

TABLE 7 Basophil Mast cell corresponding cytokine cytokine ST2L mAbhybridoma RLB* RGA^(#) release release epitope STLM15 C2244 + + + + hD1STLM62 C2494 + + + + hD1 C2519 − + + − hD3 C2521 − + + − hD3 ST2M48 NA− + nt − h/mD3 ST2M49 NA − + nt − h/mD3 ST2M50 NA − + nt − h/mD3 ST2M51NA − + nt − h/mD3 *Receptor-Ligand binding inhibition ^(#)Reporter geneassay hD1 = human ST2L D1 domain mD1 = mouse ST2L D1 domain hD3 - humanST2L D3 domain h/mD3 = human and mouse ST2L D1 and D3 domains nt = nottested

TABLE 8 Mast cell corresponding T-cell Peritoniel cytokine ST2L mAbhybridoma RLB* RGA^(#) proliferation cells lavage release** epitopeCNTO3914 C1999 + + + + + mD1 *Receptor-Ligand binding inhibition^(#)Reporter gene assay **Bone marrow derived

Example 6 ST2L Domain I Binding Antibody CNTO3914 Inhibits IntranasalIL-33-Induced Airway Hyper-Responsiveness (AHR), Airway Inflammation andMouse Mast Cell Responses

Four consecutive daily intranasal doses of 2 μg/mouse “mature” IL-33(R&D Systems) (residues 109-266 of SEQ ID NO:215) were administered tofemale BALB/c mice. Anti-mouse ST2L antibody CNTO3914 wasprophylactically dosed subcutaneously at 20 mg/kg (or 2 mg/kg or 0.2mg/kg) 24 h prior to the first IL-33 intranasal administration. Controlmice received isotype control CNTO5516 or PBS, 24 h prior to the firstIL-33 intranasal administration. Airway hyper-responsiveness (AHR) toincreasing doses of methacholine was measured using forced maneuverswith Flexivent system (Scireq, Montreal, Quebec, Canada). Formeasurement of airway hyper-responsiveness (AHR), mice were anesthetizedwith 100 mg/kg pentobarbital and 13 mg/kg phenyloin and tracheostomizedbefore connecting to FlexiVent. The mice were nebulized with saline forbaseline readings and then with two doses (10 and 20 mg/mL) ofmethacholine. For saline and each dose of methacholine, Resistance (R)values were collected for approximately 2 minutes using the “snapshot”perturbation. The peak resistance was calculated using only those valueswith a COD (coefficient of determination) above 0.9.

A separate group of mice was treated and analyzed for cellular responsein the lungs. Twenty-four hours following the last mIL-33 isotype or PBSadministration, mice were sacrificed by overdose of Sleepaway® I.P.Lungs of the mice were lavaged with 0.7 mls of cold PBS with 0.1% BSA.Resulting bronchioalveolar (BAL) fluids were centrifuged at 1200 rpm for10 minutes and the cell-free supernatants were saved at −80° C. untilanalysis of cytokine/chemokines. The BAL samples were used for totalcounts using a hemacytometer. For differential Eat counts ˜200 cellswere counted from cytospin smears after staining with wright giemsaunder light microscope.

The cell-free supernatants were collected and stored at −80° C. untilused for Luminex protein analyses. The lung tissues were removed, andthen perfused through the right ventricle using 5 mls of cold sterilePBS until adequate perfusion. The lung lobes were then placed in a FastPrep® tube containing 1 ml of PBS+protease inhibitor and frozen andstored at −80° C. for cytokine/chemokine profiling. Thecytokine/chemokine multiplex assay was performed following themanufacturer's protocol for the Murine Millipore 22-plex. Mouse mastcell protease-1 (mMCP-1) in the BAL fluid was analyzed by ELISA (MoredunScientific).

Airway Hyper-Responsiveness

CNTO3914 significantly inhibited airway hyper-responsiveness in themodel of lung inflammation induced by intranasally administered IL-33(FIG. 1). CNTO3914 was dosed subcutaneously 24 hr prior to four dailyconsecutive intranasal administrations of 2 μg/mouse mIL-33. Peak AirwayResistance as determined by Flexivent was significantly decreased with adose of CNTO3914 at 20 mg/kg. Each bar represents the mean±SEM of three(CNTO5516, an isotype control antibody) to six mice per group. Theresults have been repeated in two separate studies. Significance wasdetermined using the Two-Way ANOVA with a Bonferroni post test,CNTO3914/IL-33 **p<0.05 vs. CNTO5516/IL-33; and ***p<0.001, vs. PBS withIL-33 treatment group.

Airway Inflammation

CNTO3914 significantly inhibited Bronchoalveolar Lavage (BAL) cellrecruitment in the used model (FIG. 2). CNTO3914 was dosedsubcutaneously 24 hr prior to four daily consecutive intranasaladministrations of 2 mg/mouse mIL-33. BAL leukocytes were significantlyincreased with IL-33 administration and were significantly inhibited byCNTO3914 at 20 mg/kg. Each bar represents the mean±SEM of three(CNTO5516, an isotype control antibody) to six mice per group. Theresults have been repeated in two separate studies. Significance wasdetermined using the Two-Way ANOVA with a Bonferroni post test,***p<0.001.

Mast Cell Responses In Vivo

Mast cells store proteases including tryptases and chymases in theirgranules, which are released quickly upon mast cell activation. MouseMast Cell Protease 1 (mMCP-1) is a β chymase released by activated mastcells and known to be important for control of parasitic worm infections(Knight et al., J Exp Med 192:1849-56, 2000; Huntley et al., ParasiteImmunol 12:85-95, 1990). Measurement of mMCP-1 can be used as a markerof mast cell activation, and has been shown to be induced in a mastcell-dependent model of airway inflammation: house dust mite (Yu andChen, J Immunol 171:3808-15, 2003). MMCP-1 as determined by ELISA(Moredun Scientific) was significantly increased in BAL fluid from IL-33administered mice, and was dose-dependently inhibited by CNTO3914 (FIG.3). Significance was determined using the One-Way ANOVA with a Tukeypost test, **p<0.01, ***p<0.001, vs. IL-33 treatment.

Example 7 Anti-ST2L Domain I Binding Antibodies Inhibit Mast CellResponses In Vitro

Mast cell responses were assessed by release of chemokines and cytokinesby mouse and human mast cells as well as prostaglandin D₂ in human mastcells.

Anti-ST2L Domain I binding antibody CNTO3914 inhibited IL-33-inducedcytokine release including GM-CSF (FIG. 4A), IL-5 (FIG. 4B), and TNFα(FIG. 4C) by mouse bone marrow-derived mast cells.

Anti-human ST2L Domain I binding mab C2494 (STLM62) inhibitedIL-33-induced PGD₂ release by human cord blood-derived mast cellsinduced by 3 ng/ml IL-33 at antibody concentrations 2, 10 and 50 μg/ml(FIG. 5).

Anti-ST2L Domain I binding antibodies C2494 and C2244 inhibitedIL-33-induced GM-CSF (FIG. 6A), IL-5 (FIG. 6C), IL-8 (FIG. 6B), IL-13(FIG. 6D) and IL-10 (FIG. 6E) release by human cord blood-derived mastcells at antibody concentrations 50 μg/ml, 10 μg/ml and 2 μg/ml (FIGS. 6and 8). The degree of inhibition was dependent on cytokine/chemokinemeasured, the antibody and antibody concentration tested, and mediaused. Calculated average percent (%) inhibition was between 50.6-100% inall assays conducted at antibody concentration 2 μg/ml, and between62-100% at antibody concentration of 50 μg/ml (FIG. 9).

Anti-ST2L Domain III-binding antibodies C2521, C2519, ST2M48, ST2M49,ST2M50, and ST2M51 showed modest or no inhibition on, or stimulatedIL-33-induced cytokine release by the mast cells (FIGS. 7A, 7B, 7C, 7D,7E, 8A, 8B, 8C, 8D and 8E) at antibody concentrations 50 μg/ml and 10μg/ml. The degree of inhibition was dependent on cytokine/chemokinemeasured, the antibody tested, and media used. Calculated averagepercent (%) inhibition was between −594.4-31.9% in all assays conductedat antibody concentration 2 μg/ml, and between −481.5-36% at antibodyconcentration of 50 μg/ml (FIG. 9). In some assays, antibody ST2M50inhibited GM-CSF (FIG. 8A), IL-5 (FIG. 8C), IL-10 (FIG. 8E) and IL-13(FIG. 8D) secretion at antibody concentration 10 μg/ml.

Average % inhibition was calculated using the following formula:(1−(concentration of cytokine released in the presence of themAb)/(concentration of the same cytokine released in response to IL-33in the absence of mAb))×100. Cytokine concentrations are in pg/ml. Insome cases, the % inhibition is a negative value, indicating that thecytokine release in the presence of mAb was actually higher than thatreleased in the absence of mAb. Slight variations in the potency of themAbs may occur depending on the IL-33 concentrations used to inducecytokine release in the mast cells. Similarly, there may be slightvariations in the activity of the mAbs depending on the assay mediumused (StemPro-34 vs. RPMI/10% FCS). All tested ST2L Domain I bindingantibodies inhibited all measured cytokine and chemokine releases atleast by 50% as measured by average % inhibition at a concentration of 2μg/ml, 10 μg/ml or 50 μg/ml.

Example 8 ST2L Domain I Binding Antibodies Inhibit IntranasalIL-33-Induced Airway Remodeling

C57BL/6 mice were dose intranasally with 1 μg/mouse “mature” IL-33 (orPBS) (residues 109-266 of SEQ ID NO: 215) on days D1, D3, D5, D7, and D9and lungs were analyzed on Day 10 or Day 20. Anti-mouse ST2L antibodyCNTO3914 or isotype control (CNTO5516) was dosed subcutaneously at 2mg/kg 6 h prior to the first IL-33 intranasal administration. Controlmice received isotype control CNTO5516 or PBS, 6 h prior to the firstIL-33 intranasal administration. Inflated lungs were fixed in 10%buffered formalin for histology; stains used for analysis included H&E,Masson Trichrome and PAS.

IL-33 treatment induced moderate to marked bronchiolar epithelialhypertrophy and hyperplasia with goblet cell hyperplasia andperibronchiolar infiltrates mixed mainly with eosinophils. Bronchiolarepithelial hypertrophy and hyperplasia were not evident in the animalsreceiving CNTO3914. The Masson Trichome stains were to determine theamount of collagen present; this staining revealed goblet cellhypertrophy in IL-33 treated animals. In the animals treated withCNTO3914 infiltrates in the alveoli and peribonchiolar regions wereabsent.

Example 9 Generation of Fully Human ST2L-Antibodies

Additional human ST2L-binding Fabs were selected from de novo pIX phagedisplay libraries essentially as described in Example 3 except that thelibraries were panned using chimeric HHM-ST2L construct (SEQ ID NO: 6,Table 1) with the biotinylated antigen captured on streptavidin-coatedmagnetic beads. The phage library was blocked in PBS-T with 3% non-fatdry milk. Competitor protein, MHM-ST2L chimera (SEQ ID NO: 7, Table 1)was added to the blocking solution to drive the phage selection towardsFabs that would bind specifically to the human ST2L Domain I amino acidsequences. Phage selections were performed for three rounds followed byscreening by ELISA for Fab binding to hST2L-Fc protein.

Nineteen Fabs with binding to hST2L-Fc were isolated from theseselections and were further screened for binding to chimeric ST2Lconstructs (Table 1) as well as to the mouseST2L and humanST2L proteinsto map the domain of specificity, and characterized for their ability toblock IL-33/hST2L interaction. Fabs ST2F1, ST2F4 and ST2F6 blockedhIL-33/ST2L interaction and bound Domain I of ST2L and were movedforward into affinity maturation.

TABLE 9 HCDR1 HCDR2 HCDR3 SEQ ID SEQ ID SEQ ID Fab ID VH ID FrameworkSequence NO: Sequence NO: Sequence NO: ST2F6 ST2H41 VH3-23 SYAMS 78AISGSGGSTYYADSVKG 81 DPWSTEGSFFVLDY 84 ST2F4 ST2H39 VH3-23 SYWMH 79GISSGGGSTYYADSVKG 82 DGWGTVYFPFDY 85 ST2F1 ST2H35 VH5-51 SYWIG 80IIYPGDSDTRYSPSFQG 83 DTADFRRWDFDY 86 LCDR1 LCDR2 LCDR3 SEQ ID SEQ IDSEQ ID Fab ID VL ID Framework Sequence NO: Sequence NO: Sequence NO:ST2F6 ST2L24 Vk-L6 RASQSVDDALA 87 DASNRAT 90 QQFYNWPLT 92 ST2F4 ST2L23Vk-L6 RASQSVRDDLA 88 DASNRAT 90 QQYIHAPLT 93 ST2F1 ST2L20 Vk-B3KSSQSVLYSSNNKNYLA 89 WASTRES 91 QQSNTYPFT 94

Example 10 Affinity-Maturation of Human ST2L Binding Fabs

ST2F1, ST2F4 and ST2F6 were affinity-matured using an “in-line”maturation process described in Shi et al., j Mol Biol 397:385-396, 2010and Int. Pat. Publ. No. WO2009/085462 and Example 4. Affinity maturationlibraries were made for ST2F1, ST2F4 and ST2F6 by diversifyingcorresponding light chain libraries, B3, L6 and L6, respectively, andcombining the libraries with the Fab VH regions. The diversificationscheme for light chain residues for the L6 and B3 affinity maturationlibraries are shown in Table 10. Position numbering is according toKabat. For affinity maturation panning, biotinylated huST2-ECD-Fc wascaptured on streptavidin (SA)-coated magnetic beads at concentrations of10 nM for round 1, 1 nM for round 2, and 0.1 nM for round 3. The finalwash of round 3 was performed overnight at room temperature in thepresence of 10 nM unlabelled huST2L-ECD-Fc.

TABLE 10 Scaffold Loop Position L6 B3 L1 30 SRNAD RNDGHSY 30a — RNDGHWY30e — RNDGHSY 31 NSKD RNDGHWY 32 YWDFHSAN YNWR L2 50 ADKGYFTN YWNK L3 91RYSGF SYWH 92 RHNSL SYGN 93 NDKR STER 94 WA WYSH 96 WYFLIR YRWH

The ST2F6 light chain maturation library selections yielded improvedbinders (ST2F14, ST2F17, ST2F31 and ST2F41) (FIG. 10 and FIG. 11). Thesewere examined as Fabs using ProteOn and demonstrated modest affinityimprovements from 2 nM to 400 pM.

To further improve affinity of ST2F14, ST2F17, ST2F31 and ST2F41, thecommon heavy chain ST2H41 in ST2F14, ST2F17, ST2F31 and ST2F41 wasrandomized at HCDR1 and HCDR2Kabat positions 31, 32, 33, 35, 50, 52, 53,56 and 58 using a diversification scheme shown in Table 11. Theresulting heavy chain library was paired with the four affinity improvedlight chains ST2L32, ST2L35, ST2L49 and ST2L59, and this library waspanned and screened as described for the light chain maturationlibraries. Fabs with improved binding relative to ST2F14 were isolatedand converted to IgG for further characterization. The resultingantibodies (STLM103, STLM107, STLM108, STLM123, STLM124, STLM206,STLM207, STLM208, STLM209, STLM210, STLM211, STLM212, STLM213, STLM214,STLM215, STLM216, STLM217, STLM218, STLM219, STLM220, STLM221, STLM222)(FIG. 10 and FIG. 11) have frameworks derived from VH3-23 or Vκ-L6. Allantibodies bind ST2L Domain I and block IL-33/ST2L interaction.

TABLE 11  Position Amino Acids 31 SDNTAY 32 SDAY 33 SDAY 35 SN 50 SDNTAY52 SANTKDEGR 53 SANEY 56 SANTKDEGR 58 SDNTAY

Additional variants were designed and expressed for STLM208 VH ST2L257to replace a DP motif at the beginning of HCDR3. The sequences of thevariants are shown in FIG. 12.

Example 11 Human Framework Adaptation (HFA) of C2494

The framework adaptation process was done as essentially described inU.S. Pat. Publ. No. US2009/0118127 and Fransson et al., J Mol Biol398:214-231, 2010. Briefly, the heavy and light chain sequences werecompared with the human germline sequences (only the “01” alleles as ofOct. 1, 2007) using BLAST search against the IMGT database (Kaas, etal., Nucl. Acids. Res. 32, D208-D210, 2004; Lefranc et al., Nucl. AcidRes., 33, D593-D597, 2005). From this set of human germline genes,redundant genes (100% identical at amino acid level) and those withunpaired cysteine residues were removed. The remaining closest matchinghuman germline genes in both the framework and CDR regions were chosenas the acceptor human frameworks. A total of 9 VL and 7 VH germlinehuman frameworks were selected based upon overall sequence homology andCDR lengths as well as CDR similarity. FR-4 was selected based onsequence similarity of the IGHJ/IGJK germline genes, JK2 for the VLchains and JH1 for the VH chains (Kaas, et al., Nucl. Acid Res. 32,D208-D210, 2004; Lefranc M.-P et al., Nucl. Acid Res., 33, D593-D597,2005) with C2494 sequence). Then, the CDRs of C2494 (underlined in FIG.14) were transferred into the selected acceptor human frameworks togenerate the HFA variants, except in the region corresponding to theCDR-H1 of V_(H). For this region a combination of CDR and HV, or ashorter HCDR2 (referred to as Kabat-7, see U.S. Pat. Publ. No.US2009/0118127) were transferred from the non-human antibody into thehuman FRs because the HCDR2 residues highlighted in grey in FIG. 14 havenot been found in contact in antigen-antibody complexes of knownstructures (Almagro, J Mol. Recognit. 17, 132, 2004).

The mature protein sequence of C2494 (VL: SEQ ID NO:52; VH: SEQ ID NO:48) is shown FIG. 14. In the figure, CDR residues (Kabat) areunderlined, Chothia HV loops indicated below CDRs, and residuestransferred into selected human frameworks indicated under HVs (HFA).HCDR2 residues highlighted in grey were not transferred in all variants.

A 3D homology model for the Fv fragment of C2494 was constructed usingthe antibody modeling module of MOE (CCG, Montreal). The model wasutilized for evaluation of developability liabilities such as exposedmethionine and tryptophan residues, potential N-glycosylation anddeamidation motifs. In LCDR3, there is a potentially exposed Met (M94)residue, based upon the Fv structural model. To remove it, a variant(STLL280, O12b) with an M94L mutation was generated and characterized.For the heavy chain, the R residue in the CAR motif (Chothia residues92-94, FIG. 14) just before HCDR3 may negatively impact a cluster ofnegatively charged residues (Chothia residues D31, D32, D96 and D101a,FIG. 14), which may be important for binding. A VH with substitution ofarginine for leucine at Chothia residues 94 (CAR→CAL) was generated andcharacterized.

The mAbs combining designed heavy and light chains, together with theC2494 parents were expressed and assayed for binding to human ST2L. Fromthe generated HFA mAbs, mAbs with VH chains having IGHV1-24*01 (SEQ IDNO: 148) and IGHV1-f*01 (SEQ ID NO: 149) heavy chain frameworks (STLH195and STLH194) expressed antibodies well and bound ST2L when combined withvarious HFA light chains having IGKV3-15*01 (L2) (SEQ ID NO: 150),IGKV1-9*01 (L8) (SEQ ID NO: 151), IGKV1-5*01 (L12) (SEQ ID NO: 152),IGKV1-12*01 (L5) (SEQ ID NO: 153), IGKV1-39*01 (O12) (SEQ ID NO: 154),IGKV1-27*01 (A20) (SEQ ID NO: 155) or IGKV1-33*01 (O18) (SEQ ID NO: 156)frameworks (STLL280, STLL278, STLL277, STLL276, STLL275, STLL274,STLL273, STLL272).

Sequences of HFA VH and VL variants are shown in Table 12. Transferredresidues are underlined, and additional substitutions described abovehighlighted in grey. Table 13 shows SEQ ID NOs: as well as unique pDR(plasmid) and CBIS ID for each HFA VH and VL. Heavy and light chaincombination for generated mAbs selected for further characterization isshown in Table 14.

Table 15 shows the human frameworks (combined V and J regions) used totransfer C2494 CDRs.

TABLE 12Framework adapted VL chains (coupled to JK2 sequence). >VL2494 (parent) (SEQ ID NO: 52)ETTVTQSPASLSVATGEKVTIRCITNTDIDDVIHWYQQKPGEPPKLLISEGNTLRPGVPSRFSSSGYGTDFVFTIENTLSEDVADYYCLQSDNMLTFGAGTKLELK >VL2494-IGKV1-33*01 O18 (SEQ ID NO: 135)DIQMTQSPSSLSASVGDRVTITCITNTDIDDVIHWYQQKPGKAPKLLIYEGNTLRPGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCLQSDNMLTFGQGTKLEIK >VL2494-IGKV1-27*01 A20 (SEQ ID NO: 136)DIQMTQSPSSLSASVGDRVTITCITNTDIDDVIHWYQQKPGKVPKLLIYEGNTLRPGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLQSDNMLTFGQGTKLEIK >VL2494-IGKV1-39*01O12 (SEQ ID NO: 137)DIQMTQSPSSLSASVGDRVTITCITNTDIDDVIHWYQQKPGKAPKLLIYEGNTLRPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQSDNMLTFGQGTKLEIK >VL2494-IGKV1-12*01 L5 (SEQ ID NO: 138)DIQMTQSPSSVSASVGDRVTITCITNTDIDDVIHWYQQKPGKAPKLLIYEGNTLRPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQSDNMLTFGQGTKLEIK >VL2494-IGKV1-5*01 L12 (SEQ ID NO: 139)DIQMTQSPSTLSASVGDRVTITCITNTDIDDVIHWYQQKPGKAPKLLIYEGNTLRPGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCLQSDNMLTFGQGTKLEIK >VL2494-IGKV1-9*01 L8 (SEQ ID NO: 140)DIQLTQSPSFLSASVGDRVTITCITNTDIDDVIHWYQQKPGKAPKLLIYEGNTLRPGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQSDNMLTFGQGTKLEIK >VL2494-IGKV3-15*01 L2 (SEQ ID NO: 141)EIVMTQSPATLSVSPGERATLSCITNTDIDDVIHWYQQKPGQAPRLLIYEGNTLRPGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCLQSDNMLTFGQGTKLEIK>VL2494-IGKV1-39*01 O12b (SEQ ID NO: 142)

Framework adapted VH chains coupled to JH1>VH2494(parent) (SEQ ID NO: 48)

>VH2494-IGHV1-f*01 (SEQ ID NO: 143)

>VH2494-IGHV1-24*01 (SEQ ID NO: 144)

CDRs are underlined.

TABLE 13 SEQ HFA-variant pDR# CBIS ID ID NO: VH HFA >VH2494-IGHV1-24*019870 STLH195 144 chains >VH2494-IGHV1-f*01 9871 STLH194 143 VLHFA >VL2494-IGKV1-39*01 O12b 9865 STLL280 142 chains >VL2494-IGKV3-15*01L2 9873 STLL278 141 >VL2494-IGKV1-9*01 L8 9874 STLL277140 >VL2494-IGKV1-5*01 L12 9875 STLL276 139 >VL2494-IGKV1-12*01 L5 9876STLL275 138 >VL2494-IGKV1-39*01 O12 9877 STLL274 137 >VL2494-IGKV1-27*01A20 9878 STLL273 136 >VL2494-IGKV1-33*01 O18 9879 STLL272 135

TABLE 14 VH chains >VH2494- >VH2494- Parent* IGHV1-24*01 IGHV1-f*01 VLchains pRD# pDR4211 pDR9870 pDR9871 Parent* pDR4212 STLM126 STLM186STLM196 >VL2494-IGKV1-39*01 O12b pDR9865 STLM127 STLM187STLM197 >VL2494-IGKV3-15*01 L2 pDR9873 STLM129 STLM189STLM199 >VL2494-IGKV1-9*01 L8 pDR9874 STLM130 STLM190STLM200 >VL2494-IGKV1-5*01 L12 pDR9875 STLM131 STLM191STLM201 >VL2494-IGKV1-12*01 L5 pDR9876 STLM132 STLM192STLM202 >VL2494-IGKV1-39*01 O12 pDR9877 STLM133 STLM193STLM203 >VL2494-IGKV1-27*01 A20 pDR9878 STLM134 STLM194STLM204 >VL2494-IGKV1-33*01 O18 pDR9879 STLM135 STLM195 STLM205 *Parent= C2494 VH and VL

TABLE 15  Frameworks used for Human Framework Adaptation (HFA)Framework V Framework J SEQ region origin region origin Sequence ID NO:IGHV1-24*01 JH1 QVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMHWVRQAPGKGLEWMGGFDPE148 DGETIYAQKFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCATWGQGTLVTVSS IGHV1-f*01JH1 EVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPED 149GETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATWGQGTLVTVSS IGKV3-15*01 L2JK2 EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGI 150PARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPTFGQGTKLEIK IGKV1-9*01 L8 JK2DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASTLQSGVP 151SRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSYPTFGQGTKLEIK IGKV1-5*01 L12 JK2DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASSLESGV 152PSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSTFGQGTKLEIK IGKV1-12*01 L5 JK2DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSG 153VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPTFGQGTKLEIK IGKV1-39*01 O12 JK2DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVP 154SRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPTFGQGTKLEIK IGKV1-27*01 A20 JK2DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAASTLQSGV 155PSRFSGSGSGTDFTLTISSLQPEDVATYYCQKYNSAPTFGQGTKLEIK IGKV1-33*01 O18 JK2DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGV 156PSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPTFGQGTKLEIK

Example 12 Design of Alanine and Human Germline Mutants for ParatopeScanning

Site-directed mutagenesis was carried to assess the bindingcontributions of individual CDR residues as well as some residues havingpotential effect on other antibody characteristics. Based upon themolecular model of C2494 Fv above a subset of solvent-exposed CDRresidues were predicted to be involved in binding antigen. These weremutated to alanine and/or corresponding ‘human-like’ residue, which isthe corresponding residue in the closest matching germline gene. D101aA(Chothia residues), (D104A in SEQ ID NO: 48) substitution in C2494 VHdecreased the k_(off) about 4 fold, from 1.43×10⁻⁴ to 3.2×10⁻⁵.

As the D101aA substitution decreased of k_(off) of C2494 Fab in bindingto ST2L it was expected that the same mutation may also improve theoff-rate in the C2494 HFA variants. Thus, D101aA (Chothia numbering) wasincorporated in the VH of STLH194 (>VH2494-IGHV1-f*01, SEQ ID NO: 143)to generate a VH STLH201 (SEQ ID NO: 145). STLH201 was paired with 7light chains STLL280, STLL277, STLL276, STLL275, STLL274, STLL273 andSTLL272 (Table 13 and Table 14) to generate mAbs STLM226, STLM227,STLM228, STLM229, STLM230, STLM231 and STLM232 which were characterizedfurther. mAbs STLM226, STLM227, STLM228, STLM229, STLM230, STLM231 andSTLM232 therefore have identical LCDR1, LCDR2, LCDR3, HCDR1 and HCDR2sequences when compared to the parent C2494 antibody and a differentHCDR3 (SEQ ID NO: 146, GDFYAMAY). In addition, antibody STLM266 VLSTLM280 had a unique LCDR3: LQSDNLLT (SEQ ID NO: 147)

STLH201 (SEQ ID NO: 145):

HCDR3 incorporation D101aA (Chothia numbering) substitution:

SEQ ID NO: 146: GDFYAMAY antibody STLM266 VL STLM280 had a unique LCDR3:(SEQ ID NO: 147) LQSDNLLT

Example 13 Characterization of Anti-ST2L Antibodies

Antibodies obtained from phage display, hybridoma and human frameworkadaptation campaigns were characterized in various assays includingbinding to huST2L-ECD, cynoST2L-ECD, affinity measurements, binding tohuman/mouse chimeras to determine domain binding, receptor-ligandinhibition assay, reporter gene assays, and mast cell response assays.

Affinities of the antibodies derived from the phage display campaigns tohuman and cyno ST2L as well as their binding specificity to human ST2Lis shown in Table 16. All antibodies in Table 16 bound Domain I of humanST2L.

TABLE 16 human ST2L affinity cyno ST2L affinity ST2L-ECD KD KD domaink_(on) (M⁻¹s⁻¹) k_(off) (s⁻¹) (pM) k_(on) (M⁻¹s⁻¹) k_(off) (s⁻¹) (pM)binding STLM103 3.97E+06 1.63E−04 41 6.42E+06 2.02E−04 31 D1 STLM1072.90E+07 3.41E−04 12 1.00E+08 6.50E−04 7 D1 STLM108 2.29E+06 2.22E−04 972.05E+07 5.98E−04 29 D1 STLM123 1.37E+07 2.08E−04 15 1.00E+08 5.19E−04 5D1 STLM124 1.65E+07 7.56E−04 46 8.71E+07 2.57E−03 30 D1 STLM206 6.39E+061.60E−04 25 9.40E+07 5.83E−04 6 D1 STLM207 8.33E+06 3.95E−04 48 1.00E+082.07E−03 21 D1 STLM208 5.97E+06 6.76E−05 11 1.39E+07 7.02E−05 5 D1STLM209 6.59E+06 1.70E−04 26 3.39E+07 3.11E−04 9 D1 STLM210 1.21E+072.27E−04 19 5.70E+07 5.28E−04 9 D1 STLM211 1.70E+07 4.83E−04 29 1.00E+081.39E−03 14 D1 STLM212 1.24E+07 3.98E−04 32 1.43E+07 3.46E−04 24 D1STLM213 7.54E+06 1.08E−04 14 1.64E+07 1.24E−04 8 D1 STLM214 9.16E+062.99E−04 33 7.20E+06 2.64E−04 37 D1 STLM215 6.91E+06 1.72E−04 253.54E+07 3.69E−04 10 D1 STLM216 9.63E+06 1.58E−04 16 7.89E+07 2.64E−04 3D1 STLM217 7.27E+06 1.26E−04 17 3.81E+07 1.38E−04 4 D1 STLM218 9.89E+062.24E−04 23 1.45E+07 2.65E−04 18 D1 STLM219 7.54E+06 2.01E−04 271.07E+07 2.30E−04 22 D1 STLM220 5.80E+06 9.53E−05 16 1.60E+07 1.40E−04 9D1 STLM221 2.73E+06 9.61E−05 35 6.04E+06 1.30E−04 22 D1 STLM222 8.22E+063.01E−04 37 1.18E+07 3.45E−04 29 D1 STLM226 2.16E+07 1.93E−03 901.00E+08 3.01E−02 301 D1 STLM227 2.66E+07 1.70E−03 64 1.00E+08 2.94E−02294 D1 STLM228 2.01E+07 1.04E−03 52 1.00E+08 1.55E−02 155 D1 STLM2291.29E+07 4.45E−04 35 1.00E+08 8.50E−03 85 D1 STLM230 1.11E+07 4.26E−0438 5.06E+07 7.30E−03 144 D1 STLM231 1.97E+07 9.13E−04 46 8.27E+071.43E−02 172 D1 STLM232 1.78E+07 4.49E−04 25 1.00E+08 7.97E−03 80 D1

Affinities of the anti-ST2L antibodies from the HFA campaign in relationto the parent (STLM62, C2494) are shown in Table 17. The affinities wereanalyzed by ProteOn. The experiments were performed at 25° C. usingProteOn's PBS-T-E buffer (PBS, 0.005% P20 and 3 mM EDTA) as runningbuffer. To perform the experiments a GLC sensor chip was prepared bycovalent immobilization of goat anti-human Fc (˜5800 RUs) 122-146response units (RU) of Mab were captured. Mab capture was followed byinjection of ST2L-ECD from 0.024-15 nM (5-fold dilutions) for 4 min (200μL at 50 μL/min). The dissociation was monitored for 30 minutes for allreaction. Regeneration was performed using two 15 sec pulses of 10 mMglycine pH1.5. The data was fitted to a 1:1 with baseline drift model.

Association rates for the samples are fast, the langmuir with masstransfer model was used for curve fitting and estimation of Affinity.All of the samples had faster off rates than the parental clone andcontrol Mab. The difference in off rate was the primary contributor tothe lower affinity of the HFA variants when compared to the parentantibody.

TABLE 17 human ST2L affinity cyno ST2L affinity Sample k_(on) (M−1s−1)k_(off) (s−1) K_(D) (pM) k_(on) (M−1s−1) k_(off) (s−1) K_(D) (pM)STLM62* 1.84E+07 1.59E−04 8.67 3.84E+07 4.57E−04 12.35 STLM187 3.37E+071.59E−02 473.00 1.00E+08 1.10E−01 1100.00 STLM190 1.00E+08 5.34E−02534.00 1.00E+08 1.02E−01 1020.00 STLM191 8.46E+07 2.47E−02 292.001.00E+08 6.66E−02 666.00 STLM192 2.11E+07 8.85E−03 420.00 1.00E+089.99E−02 999.00 STLM193 4.77E+07 1.27E−02 267.00 1.00E+08 9.32E−02932.00 STLM194 1.00E+08 7.03E−02 703.00 1.00E+08 1.90E−01 1900.00STLM195 2.49E+07 6.73E−03 271.00 1.00E+08 7.19E−02 719.00 STLM1971.83E+07 1.62E−03 88.50 2.97E+07 6.88E−03 232.00 STLM199 2.17E+078.97E−04 41.40 7.78E+07 6.57E−03 84.50 STLM200 2.35E+07 1.43E−03 60.808.23E+07 1.10E−02 134.00 STLM201 1.76E+07 8.52E−04 48.40 3.55E+074.10E−03 116.00 STLM202 2.24E+07 1.19E−03 52.90 7.75E+07 1.04E−02 134.00STLM203 2.04E+07 9.67E−04 47.30 5.88E+07 6.56E−03 111.00 STLM2042.97E+07 2.41E−03 81.30 1.00E+08 2.05E−02 205.00 STLM205 1.73E+076.95E−04 40.10 4.04E+07 4.04E−03 100.00 *STLM62 = C2494, parent antibody

TABLE 18 RLB RGA Cyno Basophil IC50, IC50, endothelial cytokine OriginmAb μg/ml μg/ml assay release Phage STLM103 0.47 1.92 NT + displaySTLM107 0.44 1.10 NT ++ STLM108 0.23 2.34 ++ ++ STLM116 0.29 6.71 NT +STLM123 0.28 1.25 NT ++ STLM124 0.35 0.87 ++ ++ STLM206 0.40 0.67 ++ ++STLM207 0.36 2.30 NT ++ STLM208 0.47 0.61 ++ ++ STLM209 0.32 0.97 ++ ++STLM210 0.30 2.10 NT ++ STLM211 0.28 2.52 NT ++ STLM212 0.33 4.32 NT +STLM213 0.34 0.49 ++ ++ STLM214 0.28 2.52 NT ++ STLM215 0.29 1.30 NT ++STLM216 0.30 1.86 NT ++ STLM217 0.49 1.69 NT ++ STLM218 0.42 1.33 NT ++STLM219 0.29 3.16 NT ++ STLM220 0.39 0.60 NT ++ STLM221 0.39 2.79 NT +STLM222 0.25 1.88 NT ++ STLM226 0.26 0.25 ++ ++ STLM227 0.17 0.23 ++ ++STLM228 0.20 0.28 ++ ++ HFA STLM229 0.29 0.32 ++ ++ STLM230 0.28 0.15 ++++ STLM231 0.26 1.10 + + STLM232 0.31 0.15 ++ ++ hybridoma STLM62* 0.700.11 ++ ++ C2494 ++ strong inhibition + some inhibition − no inhibitionNT Not tested *Tested as a hybridoma RLB = Receptor-Ligand bindinginhibition RGA = Reporter gene assay

Select antibodies were tested for mast cell responses measuringinhibition of 3 ng/ml IL-33-induced IL-5, IL-13 and IL-8 release fromhuman cord blood-derived mast cells as described using 100 μg/ml, 10μg/ml, 1 μg/ml, 0.1 μg/ml or 0.01 μg/ml antibody in RPMI+10% FCS. Inthese assay conditions, all antibodies tested inhibited IL-33-inducedIL-5, IL-13 and IL-8 cytokine release by about 40%-100% at an antibodyconcentration 100 μg/ml when compared to a control sample induced withIL-33.

Example 14 Anti-ST2L Antibody Inhibits Downstream Signaling Pathways inHuman Basophils

Anti-ST2L antibodies were tested for their ability to inhibit p38 MAPKsignaling in human basophils.

Whole blood was collected in heparinized tubes and brought to roomtemperature (RT) prior to initiation of the assay. 1 mL of blood wasaliquotted into 50 mL conical tubes and either anti-ST2L antibody(STLB252) or isotype control (CNTO 8937) diluted in PBS was added for afinal concentration of 2, 20, or 200 μg/mL. Tubes were swirled gently tomix and placed in incubator at 37° C.×30 minutes, swirling gently after15 minutes. Blood was then stained with fluorochrome-labeled antibodiesagainst cell surface antigens (CD123-FITC, CRTH2-PCP-CY5.5, andCD45-APC-C7) and tubes were incubated at 37° C. for 15 minutes. 1 mL ofwarmed culture media (RPMI-1640/10% FBS/1% pen-strep) was added to eachtube before IL-33 diluted in warmed culture media was added for a finalconcentration of 10 ng/mL. Samples were incubated at 37° C.×10 minutesprior to the addition of 20 mLs of pre-warmed BD Phosflow Lyse/Fixbuffer to each tube, in order to simultaneously lyse the red blood cellsand fix the samples. Tubes were mixed well by inverting 10 times andincubated at 37° C.×10 minutes. Samples were washed with 20 mLs sterileRT PBS, resuspended in 2 mLs of 1×RT BD Perm/Wash Buffer, and incubatedat RT×30 minutes. Samples were washed once with 2 mLs BD Perm/Washbuffer and then resuspended in 400 μL BD Perm/Wash buffer. PE-labelledantibody against intracellular p38-MAPK (vCell Signaling, Cat. 6908S)was added and samples were incubated 30 min at RT, protected from light.Samples were washed once with 5 mLs Perm/Wash buffer before beingresuspended in 100 μL FACS buffer and transferred to a 96-wellround-bottom plate. Samples were analyzed using a BD LSRII FlowCytometer utilizing a high-throughput system (HTS) collecting as manyevents as possible for each sample. Data was analyzed using FloJosoftware. Basophils were identified as CD45⁺CRTH2⁺CD123⁺ and the percentof p38 MAPK positive basophils was assessed for each condition.Pre-incubation of whole blood with anti-ST2L mAB (STLB252) resulted in adose-dependent inhibition of IL-33 induced p38-MAPK phosphorylation,whereas no effect was seen with isotype control (CNTO 8937). Theanti-human ST2L antibody specifically blocked basophil activation byrecombinant human IL-33 in the context of whole blood. The resultssuggest that anti-ST2L antibodies inhibit signaling by endogenous IL-33in vivo.

TABLE 19 Isotype IL-33 STLB252 control % phosphorylated (10 ng/ml)(μg/mL) (μg/mL) p38 MAPK − 0 0 2.2 + 0 0 80.6 + 2 0 44.4 + 20 0 15.7 +200 0 1.2 + 0 2 76.7 + 0 20 79 + 0 200 77

Example 15 In Vivo Target Engagement by Anti-ST2L Antibody

Intranasal mIL-33 6 Hour In Vivo Model of BAL Cell Recruitment

A single dose of 1.2 μg/mouse mIL-33 (R&D systems #3626-ML/CF) or PBSwas administered to male Balb/c mice (6-8 weeks old, Taconic). Ratanti-mouse ST2L antibody CNTO 3914 or at 2, 0.2, 0.06, or 0.02 mg/kg, 24hrs prior to the first mIL-33 intranasal administration. Isotype control(ITC) mAb CNTO 5516 was dosed subcutaneously at 2 mg/kg. Six hoursfollowing the mIL-33 (or PBS) administration, mice were sacrificed andblood was collected for serum analysis. Bronchoalveolar lavages (BAL)were performed by injecting two volumes of 0.7 mL of PBS/0.1% BSA intothe lungs and retrieving the effluent. The BALs were centrifuged (1200rpm, 10 minutes) and the cell pellet was resuspended in 200 μl PBS fortotal and differential cell counts using a hemacytometer (onWright's—Giemsa-stained cytospin preparations).

Measurement of CNTO 3914 in Mouse Serum

MSD SA-STD plates were blocked with 50 μL per well of assay buffer for 5minutes. The plates were turned over to remove assay buffer and tappedon paper towels. 50 μl per well of 1.4 μg/mL biotinylated recombinantmouse ST2L/IL1R4/Fc chimera (R&D System) in assay buffer were added andincubated overnight in the refrigerator. 150 μL of assay buffer wasadded to each well of the pre-coated plates without removing the coatingreagent and incubated for 30 minutes. The plates were washed three timeswith wash buffer on the plate washer. The plates were tapped lightly onpaper towels to remove residual wash buffer. 50 μL per well of CNTO 3914sample was added to each well of the plate. The plate was incubated forone hour with gentle vortexing at ambient temp. The plates were washedthree times with wash buffer on the plate washer. 50 μL per well oftitration of ruthenium-labeled mouse anti-mouse IgG1b (BD Biosciences)was added to each well of the plate. The plate was incubated for onehour with gentle vortexing at ambient temp. The plates were washed threetimes with wash buffer on the plate washer. 150 μL of read buffer wereadded to each well of the plate. The plates were immediately read on theMSD sector imager 6000 Reader for luminescence levels.

Whole Blood Assay

Blood was diluted 1:4 in DMEM media+1% Penicillin+streptomycinsolution+/−10 ng/ml mouse IL-33 in Sarstedt filter tubes. The tubes wereincubated at 37° C. overnight, then cytokine and chemokine levels weremeasured on the supernatants using the Millipore Milliplex MouseCytokine/Chemokine Kit according to manufacturer's instructions.

Results

Anti-ST2L antibody was detectable in the serum of mice 24 hourspost-dosing with 0.2 or 2 mg/kg CNTO 3914 (FIG. 16A).

Intranasal administration of IL-33 induced cell recruitment to theairways at 6 h (FIG. 17B). Anti-ST2L mAb administration reduced BAL cellrecruitment; 0.2 mg/kg was the minimum dose needed to see significantinhibition of BAL cell recruitment (FIG. 16B). Statistical significancewas calculated using One-way ANOVA.

Whole blood stimulated with mouse IL-33 showed increased levels ofcytokine and chemokines, including IL-6 (FIG. 16C) and MCP-1 (FIG. 17D),after 24 h. In mice dosed with 20 mg/kg or 2 mg/kg anti-ST2L mAb CNTO3914, IL-6 and MCP-1 levels were reduced compared to CNTO5516 (isotypiccontrol anti-mouse IgG1), implying target engagement. The minimum dosethat correlated with inhibition in the whole blood assay, 2 mg/kg, alsoinhibited BAL cell recruitment (FIG. 16B).

Collectively this data confirms that the anti-ST2L mAb reaches site ofaction and the intended pharmacologic effect was accomplished (impliestarget engagement).

Example 16 Epitopes of Anti-ST2L Antibodies

Epitope mapping and competition studies were conducted to selectanti-ST2L antibodies.

Competition Binding Assays

Competition binding assays were performed to evaluate different bindingepitope groups for anti-ST2L mAbs. 5 μl (10 μg/ml) of ST2L-ECD proteinwas coated on MSD HighBind plate (Meso Scale Discovery, Gaithersburg,Md.) per well for 2 hr at room temperature. One-hundred and fiftymicroliters of 5% MSD Blocker A buffer (Meso Scale Discovery,Gaithersburg, Md.) was added to each well and incubated for 2 hr at roomtemperature. Plates were washed three times with 0.1 M HEPES buffer, pH7.4, followed by the addition of the mixture of the MSD fluorescence dye(sulfo tag, NHS ester) labeled individual anti-ST2L antibody withdifferent competitors. Labeled antibody, 10 or 30 nM, was incubated withincreasing concentrations of competitor antibodies, from 1 nM to 2 or 5μM, and then added to the designated wells in a volume of 25 μL mixture.After 2-hour incubation with gentle shaking at room temperature, plateswere washed 3 times with 0.1M HEPES buffer (pH 7.4). MSD Read Buffer Twas diluted with distilled water (4-fold) and dispensed at a volume of150 μL/well and analyzed with a SECTOR Imager 6000.

Following antibodies were used in competition assays: ST2L Domain Ibinding neutralizing antibodies STLM208, STLM213, C2244 (STLM15) andC2494 (STLM62), ST2L Domain III binding antibody C2539, and anon-neutralizing anti-ST2L antibody C2240 binding Domain I of humanST2L. FIGS. 17A and 18B shows the competition experiments. Based on theexperiment, the epitope bins identified were: BinA: mAbs C2244, C2494,STLM208 or STLM213; BinB: mAb C2240, BinC: C2539. The antibodiesblocking IL33/ST2L interaction and inhibiting mast cell responses werefound in the same epitope bin and to cross-compete with each other.Summary of the competition data is shown in Table 20.

TABLE 20 Labeled Antibody Competitor C2240 C2539 C2244 C2494 C2240 + − −− C2539 − + − − C2244 − − + + C2494 − − + + STLM208 − − + + STLM213 −− + +Epitope Mapping: H/D Exchange Analysis

For H/D exchange, the procedure used to analyze the antibodyperturbation are similar to the one described previously (Hamuro, Y., etal., Journal of Biomolecular Techniques, 14:171-182, 2003; Horn, J. R.,et al., Biochemistry, 45: 8488-8498, 2006) with some modification.Recombinant ST2-ECD (expressed from HEK293E with C-terminal His-tag)(residues 18-328 of SEQ ID NO: 157) was incubated in a deuterated watersolution for pre-determined times resulting in deuterium incorporationat exchangeable hydrogen atoms. The deuterated ST2-ECD was captured on acolumn containing immobilized anti-ST2L C2244 Fab molecules and thenwashed with aqueous buffer. The back-exchanged ST2-ECD protein waseluted from the column and localization of deuterium containingfragments was determined by protease digestion and mass spec analysis.

FIG. 18 shows a simplified H/D exchange map of the human ST2-ECD(soluble ST2) complexed with C2244 Fab. Residues 18-31 of ST2-ECD of SEQID NO: 119 (amino acid residues RCPRQGKPSYTVDW; SEQ ID NO: 210) wereprotected by the Fab (corresponding to residues 35-48 of full lengthST2L of SEQ ID NO: 1. The data indicates that C2244 binds to epitopeRCPRQGKPSYTVDW; SEQ ID NO: 210), and that antibodies competing withC2244 (C2494, STLM208 or STLM213) are likely to bind the same oroverlapping epitope.

Epitope Mapping by Mutagenesis

Several ST2L mutants were generated having substitutions tocorresponding mouse residues at ST2L Domain I. The tested antibodies donot cross-react with mouse ST2L, therefore it is expected that ST2Lvariants with abolished and/or reduced binding are indicative of epitoperesidues at the substitution sites on ST2L. Variants were made intoconstruct HH-ST2L having residues 19-205 of full length ST2L of SEQ IDNO: 1 using standard methods. Antibodies were tested for binding to theST2L variants by ELISA or Proteon.

Surface Plasmon Resonance

Binding studies were performed using the ProteOn XPR36 ProteinInteraction Array system (Bio-Rad) (Bravman T, et al. Anal Biochem358:281-288, 2006). Anti-human/anti-mouse Fc mixture (JacksonImmunoResearch, Cat#, 109-005-098/115-005-071) was immobilized on theGLC sensor chip by amine-coupling chemistry. Individual anti-ST2L mAbwas then captured by flowing (1 μg/mL) antibody solution prepared in PBScontaining 0.5% Nonidet P-40 and 0.5% Na-deoxycholate). The signal inthe surfaces reached ˜250 resonance units (RU, 1 RU=1 pg protein/mm²) inthe anti-Fc-coated surfaces, confirming that these antibodiesspecifically capture anti-ST2L mAbs. After 90° rotation of the fluidsystem, wild type of ST2L-D1D2 or variant proteins (0.5 mg/mL in PBScontaining 0.5% Nonidet P-40 and 0.5% Na-deoxycholate) was injected inthe parallel flow channels. All of these assays were performed at 25° C.The ST2L-D1D2-dependent signals on the surfaces were obtained by doublereferencing, subtracting the response observed on surfaces immobilizingthe antibodies alone, and the signal observed injecting the vehiclealone (which allows correction for binding-independent responses). Theresulting sensorgrams were fitted by the simplest 1:1 interaction model(ProteOn analysis software), to obtain the corresponding association anddissociation rate constants (k_(a) and k_(d)).

FIG. 19 shows the ST2L variants that were made and affinity of ST2B206and ST2B252 anti-ST2L antibodies to the variants. Variant 93NL94(substitution 93TF94→93NL94) reduced binding affinity of both STLM208and STLB252 by about 5-fold from about 10.8×10⁻¹² M to about 49.5×10⁻¹²M. Lack of significant reduction of binding affinity implies that thebinding energy for the interaction between antibody and ST2L-D1D2 is asum of epitope region (RCPRQGKPSYTVDW; SEQ ID NO: 210) identified by H/Dexchange analysis and additional contribution from this 93NL94 site.Residue numbering is according to full length human ST2L of SEQ ID NO:1.

Example 17 ST2L Domain I Binding Antibodies Inhibit Primary Human LungMast Cell Responses In Vitro

Ability of the ST2L Domain I binding antibodies to inhibit lung mastcell responses were assessed by release of chemokines and cytokines inprimary human lung mast cells.

Isolation of Primary Human Lung Mast Cells

Primary human lung mast cells were isolated from normal non-smokertissue obtained from the International Institute for the Advancement ofMedicine. Cells were dispersed from the lung parenchyma and smallairways by mincing, washing, and digesting the parenchyma tissueovernight at 37° C. in collagenase and hyaluronidase enzymes. Cells werecollected, washed, and subjected to an enrichment procedure using theCD117 MicroBead Kit (human) from MACS Miletnyi Biotec to positivelyselect the mast cells from the population. Prior to experimentation,mast cells were cultured for 6 weeks in StemPro-34+200 ng/ml stem cellfactor. Two weeks after isolation, cells were phenotypicallycharacterized using flow cytometry to determine the percent mast cellpurity. The cells used in subsequent assays were 89% double positive forCD117 (C-kit or stem cell factor receptor) and Fc£R1 (the high affinityIgE receptor). Furthermore, they were 94.2% positive for ST2L; therebyconfirming their mast cell phenotype.

Cytokine Release Assay from Primary Human Lung Mast Cells

Primary human lung mast cells that had been cultured in StemPro-34+200ng/ml stem cell factor for approximately 6 weeks were collected, andwashed by centrifugation in RPMI (10% heat-inactivated FCS). Cells werecounted and plated in RPMI/10% FCS medium at a density of 65,000 cellsin a 96 well plate. The Anti-ST2L Domain I binding Mabs were added tothe primary lung mast cells, and allowed to bind for 30 minutes at 37°C. prior to stimulation with IL-33. Cells were stimulated for 24 hourswith 3 ng/ml IL-33 in order to initiate accumulation of variousmediators into the culture supernatant. Culture supernatant washarvested and stored frozen until assaying in a custom Milliplex 9-plexkit.

Anti-ST2L Domain I binding antibody, STLM208, inhibited IL-33-inducedGM-CSF (FIG. 20A), IL-5 (FIG. 20B), IL-8 (FIG. 20C), and IL-13 (FIG.20D) release in primary human lung mast cells at antibody concentrations100 μg/ml, 10 μg/ml and 1 μg/ml. Similar results were obtained using thecord blood-derived mast cells (data not shown).

We claim:
 1. An isolated human or human-adapted antibody antagonist orfragment thereof that specifically binds Domain I (SEQ ID NO: 9) ofhuman interleukin-1 receptor like 1 (ST2L) and blocks interleukin-33(IL-33)/ST2L interaction, comprising a heavy chain variable region (VH),a light chain variable region (VL), a heavy chain complementarity region(HCDR) 1 (HCDR1), 2 (HCDR2), 3 (HCDR3), a light chain complementarityregion (LCDR) 1 (LCDR1), 2 (LCDR2) and 3 (LCDR3), wherein a. the HCDR1comprises the amino acid sequence of SEQ ID NO: 97; b. the HCDR2comprises the amino acid sequence of SEQ ID NO: 114; c. the HCDR3comprises the amino acid sequence of SEQ ID NO: 84; d. the LCDR1comprises the amino acid sequence of SEQ ID NO: 130; e. the LCDR2comprises the amino acid sequence of SEQ ID NO: 90; and f. the LCDR3comprises the amino acid sequence of SEQ ID NO:
 134. 2. The isolatedantibody of claim 1 comprising the VH of SEQ ID NO:
 191. 3. The isolatedantibody of claim 2 comprising the VL of SEQ ID NO
 209. 4. The isolatedantibody of claim 1, comprising the VH of SEQ ID NO: 191 and the VL ofSEQ ID NO: 209.